Methods for treating hcv

ABSTRACT

This invention folates to combinations of therapeutic molecules useful for treating hepatitis C virus infection. The present invention relates to methods, uses, dosing regimens, and compositions,

PRIORITY OF INVENTION

This application claims priority to U.S. Provisional Application No. 81/535,885, filed 18 Sep. 2011; and to U.S. Provisional Application No. 51/581,753, filed 18 Nov. 2011. The entire content of each of these provisional applications is hereby incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to combinations of therapeutic molecules useful for treating hepatitis C virus infection. The present invention relates to methods, uses, dosing regimens, and compositions.

BACKGROUND OF THE INVENTION

Hepatitis is a disease occurring throughout the world. Hepatitis is generally of viral nature, although, if considered a state of chronic inflammation of the liver, there are other known, non-infectious causes. Viral hepatitis is by far the most common form of hepatitis. The U.S. Centers for Disease Control has estimated that at least 1.8% of the U.S. population has serologic evidence of HCV infection, in the majority of cases associated with chronic active infection. HCV is a positive-stranded RNA virus belonging to the Flaviviridae family and has closest relationship to the pestiviruses that include hog cholera virus and bovine viral diarrhea virus.

The HCV genome is a single-stranded, positive-sense RNA of about 9,600 bp coding for a polyprotein of 3009-3030 amino acids, which is cleaved co- and pest-translationally by cellular and two viral proteinases info mature viral proteins (core, E1, E2, p7, N52, NS3, NS4A, NS4B, NS5A, NS5B). The structural proteins, E1 and E2, are believed to be embedded into a viral lipid envelope and form stable heterodimers. The structural core protein is believed to Interact with the viral RNA genome to form the nucleocapsid. The nonstructural proteins designated NS2 to NS5 include proteins with enzymatic functions involved in virus replication and protein processing including a polymerase, protease, and helicase. HCV replicates through the production of a complementary negative-strand RNA template.

HCV is a genetically diverse virus. Within a single infected patient, many variant viruses can be identified, leading to the description ‘viral swarm’, or viral quasispecies. Within the global human population, HCV is also genetically diverse, with at least 8 major ‘genotypes’ identified (Genotypes 1-6), and numerous subtypes (i.e., HCV Genotype 1a and 1b). HCV genotypes are defined by genomic phylogenetic analysis, and diagnosed (in a givers patient) by HCV RNA sequence-based diagnostic assays.

The main route of infection with HCV is blood exposure. The magnitude of the HCV infection as a health problem is illustrated by the prevalence among high-risk groups. For example, in some surveys, 60% to 90% of hemophiliacs and more titan 80% of intravenous drug abusers in western countries had chronic HCV infection. For intravenous drug abusers, tie prevalence varies from about 28% to 80% depending on the population studied. The proportion of tow HCV infections associated with blood or blood product transfusion has been markedly reduced due to pharmaceutical advances and widespread use of sensitive serologic and RNA detection assays used to screen blood donors, however, a large cohort of aging, chronically infected persons is steeds established.

One available treatment for HCV infection is pegylated interferon-α (PEG-IFN α1a or PEG-IFN α1b), which is, under current treatment guidelines, administered weekly by subcutaneous injection for 24 to 48 weeks, dependent upon the HCV viral genotype being treated. Although greater than 50% of patients with Genotype 1 HCV infection may be expected to have suppression of HCV viremia at the completion of 48 weeks therapy, a significant proportion of these patients will have viral relapse. Accordingly, a Sustained Virologic Response (SVR, defined as HCV RNA negativity 24 weeks post treatment cessation, and considered tantamount to ‘cure’) is only achieved in 30-40% of Genotype 1 HCV infections treated with PEG-IFN alone. In addition, treatment with PEG-IFN+RBV is not well tolerated, with an adverse event profile that includes flu-like symptoms, thrombocytopenia, anemia, and serious psychiatric side effects. While treatment with the current standard of care is suboptimal, many patients are precluded from ever starting therapy due to comorbidities common in HCV-infected populations, including psychiatric disorders, advanced liver disease, end substance abuse.

Ribavirin is a nucleoside analog antiviral drug. Ribavirin is typically taken orally (by mouth) twice a day. The exact mechanism for ribavirin is unknown. However, it is believed that when ribavirin enters a cell it is phosphorated; it then acts as an inhibitor of inosine 5′-monophosphate dehydrogenase (IMPDH). IMPDH inhibitors such as ribavirin reduce the intracellular synthesis and storage of guanine, a nucleotide “building block” necessary for DMA and RNA production, thus inhibiting viral replication. IMPDH inhibitors also interfere with the reproduction of rapidly proliferating cells and cells with a high rate of protein turnover. Treatment with ribavirin monotherapy has little effect on HCV RNA levels, but is associated with a decline in serum alanine transferase (ALT). This observation suggests that ribavirin may not be acting as an antiviral agent, but rather as a modulator of immune system function. Ribavirin is only approved for use, for HCV infection, in combination with IFN.

Treatment with the combination of PEG-IFN plus ribavirin improves SVR rates over those observed with PEG-IFN alone, in large part due to reduction in the frequency of viral relapse at the cessation of therapy. Large clinical trial SVR rates for PEG-IFN/ribavirin trusted patients with HCV Genotype 1 infection have ranged from 40-55%. At the present time, PEG-IFN/ribavirin therapy is considered the ‘standard-of-care’ treatment for chronic HCV infection. The standard of care is, however, expected to change rapidly in the near future with approval of direct acting antiviral agents which will, initially, be used in combination with PEG-IFN/ribavirin. Unfortunately, different genotypes of HCV respond differently to PEG-IFN/ribavirin therapy; for example, HCV genotype 1 is more resistant to therapy than types 2 and 3. Additionally, many current treatments for HCV produce unwanted side effects. Thus, there is currently a need for new anti-viral therapies. In particular there is a need for new antiviral therapies that produce fewer unwanted side-effects, that are more effective against a range of HCV genotypes, or that have less complicated dosing schedules, i.e. that require administration of agents fewer times during a day.

SUMMARY OF THE INVENTION

The present invention presides compositions and therapeutic methods that are useful for treating viral infections (e.g. HCV). Certain compositions and methods of the invention produce fewer unwanted side-effects, are more effective against a range of HCV genotypes, reduce the potential for viral rebound due to resistance selection and have shortened less complicated dosing schedules than currently available therapies,

Accordingly, in one embodiment the invention provides a composition comprising two or mere compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 18 and pharmaceutically acceptable salts thereof.

In another embodiment the invention provides a method of treating an HCV infection in a human, comprising administering two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and pharmaceutical acceptable salts thereof to the human.

In another embodiment the invention provides a method for ameliorating one or more symptoms of an HCV infection in a human, comprising administering two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and pharmaceutically acceptable salts thereof to the human.

In another embodiment the invention provides a method for reducing viral load in e human with HCV, comprising administering two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 18 and Compound 18 and pharmaceutically acceptable salts thereof to the human.

In another embodiment the invention provides a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human, comprising administering two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 8, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound and Compound 18 and pharmaceutically acceptable salts thereof to the human.

In another embodiment the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and pharmaceutically acceptable salts thereof in medical therapy.

In another embodiment the invention provided the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and pharmaceutically acceptable salts thereof for the prophylactic or therapeutic treatment of a viral (e.g. HCV) infection.

In another embodiment the invention provides the use of a composition of the inve

for the prophylactic or therapeutic treatment of a viral (e.g. HCV) infection.

In another embodiment the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 18 and pharmaceutically acceptable salts thereof to prepare a medicament for treating a viral (e.g. HCV) infection

human.

In another embodiment the invention provides the use of a composition of the inve

to prepare a medicament for treating a viral (e.g. HCV) infection in a human.

In another embodiment the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 18 and pharmaceutically acceptable salts thereof to prepare a medicament for ameliorating one or more symptoms viral (e.g. HCV) infection in a human.

In another embodiment the invention provides the use of a compositors of the inver

to prepare a medicament for ameliorating one or more symptoms of a viral (HCV) infection human.

In another embodiment the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and pharmaceutically acceptable salts thereof to prepare a medicament for reducing viral load in a human.

In another embodiment the invention provides the use of a composition of the invention to prepare a medicament for reducing viral feed in a human.

In another embodiment the invention provides the us(c) of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and pharmaceutically acceptable salts thereof to prepare a medicament for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human.

In another embodiment the invention provides the use of a composition of the invention to prepare a medicament for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human.

The compositions and methods of the invention may provide “synergy” and “synergistic effects”, i.e. the effect achieved when the active ingredients (including two or more Combination Compounds) are used together is greater than the sum of the affects that results from using the compounds separately.

The compositions and methods of the invention are beneficial because they provide treatments for a wide range of HCV genotypes and because they cause fewer or lass serious side effects than current HCV therapies (e.g. treatments that include the administration of interferon). Additionally, certain combinations of compounds (e.g. Compounds 10 and 5, Compounds 10 and 6, and Compounds 10, 5, and 6) may provide a Sustained Virological Response (SVR) that is a significantly higher than that achieved by current therapies (e.g. HCV therapies). For example, some combinations of compounds may provide an SVR that is at least about 70% or at least about 80%.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings. The fact that a particular term or phrase is not specifically defined should not be correlated to indefiniteness or lacking clarify, but rather terms herein are used within their ordinary meaning. When trade names are used herein, applicants intend to independently include the trade name product and the active pharmaceutical ingredient(s) of the trade name product.

As used herein the term “Combination Compounds” refers to Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

As used herein, Compound 1 is:

Compound 1 may also be referred to as 5-((6-(2,4-bis(trifluoromethyl)phenyl)pyridazin-3-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine, 5-[[6-[2,4-bis (trifluoromethyl)phenyl]pyridazin=3-yl]methyl]-2-(2-fluorophenyl).

As used herein, Compound 2 is:

Compound 2 may also be referred to as (2R, 6S, 13aR, 14aS, 16aS)-2-(8-chloro-2-(2-(isopropylamine)thiazol-4-yl)-7-methoxyquinolin-4-yloxy)-6-(cyclopentyloxycarbonylamino)-5,16-dioxooctadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-14a-yl(2,6-diflurobenzyl)phosphinic acid.

As used herein, Compound 3 is:

As used herein, Compound 4 is:

As used herein, Compound 5 is:

As used herein, Compound 5 is:

As used herein, Compound 7 is:

As used herein, Compound 8 is:

As used herein Compound 9 (diastereomer at P) is:

With regard to Compound 9, reference is made to U.S. Pat. No. 7,984,580 and US 2010/029 (both of which ere incorporated by reference) with regard to manufacture and purification

Compound 9.

As used herein, Compound 10 (S-isomer of Compound 9) is:

With regard to Compound 10, reference is made to U.S. Pat. No. 7,984,580 and US 2010/0298257, (both of which are incorporated by reference) with regard to manufacture a purification of Compound 10.

As used herein, Compound 11 is:

With regard to Compound 11, reference is made to US 2010/0081828 (which is her incorporated by reference) with regard to manufacture and purification of Compound 11.

As used herein, Compound 12 (diastereomer at P) is:

With regard to Compound 12, reference is made to US 20110015146 (which is hereby incorporated by reference) with regard to manufacture and purification of Compound 12.

As used herein, Compound 13 (S-diastereomer of Compound 12 at P) is:

With regard to Compound 13, reference is made to US 20110015146 (which is here incorporated by reference) with regard to manufacture and purification of Compound 13.

As used herein, Compound 14 is:

With regard to Compound 14, reference is made to U.S. Pat. No. 7,964,580 (which is hereby incorporated by reference) with regard to manufacture and purification of Compound 14.

As used herein, Compound 15 is:

With regard to Compound 15, reference is made to U.S. Pat. No. 7,964,580 (which is hereby incorporated by reference) with regard to manufacture and purification of Compound 15.

As used herein, Compound 16 is:

With regard to Compound 16, reference is made to U.S. Pat. No. 7,429,572 (which is hereby incorporated by reference) with regard to manufacture and purification of Compound 18.

With regard to ribavirin, reference is made to EP 0 093 401 B1, herein incorporated by reference with regard to a process for manufacture as well as to nomenclature concerning ribavirin. As used herein, ribavirin refers to:

Ribavirin is also referred to as 1-βD-ribofuranoayl-¹H-1,2,4-Triazole-3-carboxamide, 1-β-D-ribofuranosyl-1,2,4-trizol-3-carboxyamide; 1-β-D-Ribofuranosyl-1,2,4-trizole-3-carboxamide; COPEGUS (Roche); DRG-0082g; HSPB 6513; ICN 1229; MegaRibavirin (e.g. in formulations of 100 mg of ribavirin/mL); NSC 163039; RAVANEX (BioPartners); REBETOL (Schering-Plough; Aesca; Bayer Schering Pharma; Essex; Pfizer; Trading Pharma; Zuellig Pharma); Ribamide; RIBAMIDIL (Biopharma, Russia); RIBASPHERE (Three Rivers Pharmaceuticals); Ribavarin; Ribavirina; Tribavirin; VILONA (Valeant Pharmaceuticals; ICN Pharmaceuticals); VIRAMID (ICN Pharmaceuticals; Alfa Wassermann); VIRAZOLE (Valeant Pharmaceuticals); and VIRIZADOLE (Uci-farma, Sao Bernardo do Campo, Sao Paulo, Brazil). In addition, as used herein ribavirin includes analogs of ribavirin, including tarlbavirin (VIRAMIDINE, ICN 3142).

The term “interferon” includes 1) interferons, e.g., pegylated rIFN-alpha 2b (PEG-Intron, Merck & Co., Inc.), pegylated rIFN-alpha 2a (PEGASYS, Hoffmann-La Roche Inc.), rIFN-alpha 2b (INTRON® A, Merck & Co., Inc.), rIFN-alpha 2a (Roferon®-A, Hoffmann-La Roche Inc.), interferon alpha (MULTIFERON® Viranative AB Corporation, OPC-18, Alfaferone, Alfanative, subalin), interferon alfacon-1 (Valeant), interferon alpha-n1 (Wellferon™, Glaxo Wellcome), interferon alpha-n3 (ALFERON®-Hemispherx Biopharma, Inc.), interferon-beta-1a (AVONEX® Biogen Idec, DL-8234 Daiichi Pharmaceutical Co. Ltd), inferon-omega (omega DUROS®, Alza Corporation, Intarcia Therapeutics, Inc.; Biomed 510, Intarcia Therapeutics, Inc.), albinterferon alpha-2b (ALBUFERON®, Human Genoma Sciences, INC.), IFM alpha-2b XL, BLX-883 (LOCTERON®, Biolex Therapeutics, INC.), DA-3021, glycosylated interferon alpha-2b (AVI-005), PEG-INFERGEN®, Amgen, Inc., Pegylated Interferon lambda-1(type III) (PEGylated IL-29), and BELEROFON®, Nautilus Biotech.

The term “combination therapy” means compositions or methods or uses or the like that incorporate two or more of the Combination Compounds. Combination therapy may also incorporate other active ingredients in addition to the two or more of the Combination Compounds including, but not limited to: ribavirin, an interferon, an alpha-glucosidase 1 inhibitor, a hepatoprofectant, a Toll-like receptor (TLR)-7 agonist, a cyclophilin inhibitor, an HCV viral entry inhibitor, an HCV maturation inhibitor, and an HCV IRES inhibitor.

The term “active ingredient” means a component of a combination therapy that a exerts or is capable of exerting a pharmaceutical effect including any of the Combination Compounds, ribavirin, an interferon, an alpha-glucosidase 1 inhibitor, a hepatoprofectant, a TLR-7 agonist, a cyclophilin inhibitor, an HCV viral entry inhibitor, an HCV maturation inhibitor, and an HCV IRES inhibitor.

The term “treating” and grammatical equivalents thereof, when used in the context of treating a disease, means slowing or stepping the progression of a disease, or ameliorating at least one symptom of a disease, more preferably ameliorating more than one symptom of a disease. For example, an HCV patient may experience an improvement in one or all of the following symptoms that can be associated with HCV infection: increase in alanine aminotransferase (ALT) levels, fever, headache, muscle aches, jaundice, fatigue, loss of appetite, nausea, vomiting and diarrhea. Treatment of a hepatitis C virus infection can include reducing the HCV viral load in an HCV infected human being.

Certain of the compounds described herein contain one or more chiral canters, or may otherwise be capable of existing as multiple stereoisomers. The scope of the present invention includes mixtures of stereoisomers as well as purified anantiomers or enantiomerically/diastereomerically enriched mixtures. Also included within the scope of the invention ere the individual isomers of the compounds represented by the formulae shown herein, as well as any wholly or partially equilibrated mixtures thereof. The present invention also includes the individual isomers of the compounds represented by the formula shown herein as mixtures with isomers thereof in which one or more chiral centers are inverted. Stereochemical definitions and conventions used herein generally follow S, P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York, herein incorporated by reference in its entirety.

Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (−) are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) or l meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory.

A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often celled an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

Combinations

The present invention encompasses combinations of two or more of the Combination Compounds. Table I showing possible two-way (Combinations 1-21), three-way (Combinations 22-58), four-way (Combinations 57-92) and five-way (Combinations 93-113) combinations of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 of the invention is provided below. Compound 4, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 are nucleoside inhibitors of HCV NS5b polymerase and combinations of Combination Compounds will most often include only one of Compound 4, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 (See Column 6 of Table I).

Compound 4 Or Compound 9 Or Compound 10 Or Compound 11 Or Compound 12 Or Compound 13 Or Compound 14 Or Compound 15 Compound Compound Compound Or Compound Compound Compound 1 2 3 Compound 16 5 6 7 Combination 1 X X Combination 2 X X Combination 3 X X Combination 4 X X Combination 5 X X Combination 6 X X Combination 7 X X Combination 8 X X Combination 9 X X Combination 10 X X Combination 11 X X Combination 12 X X Combination 13 X X Combination 14 X X Combination 15 X X Combination 16 X X Combination 17 X X Combination 18 X X Combination 19 X X Combination 20 X X Combination 21 X X Combination 22 X X X Combination 23 X X X Combination 24 X X X Combination 25 X X X Combination 26 X X X Combination 27 X X X Combination 28 X X X Combination 29 X X X Combination 30 X X X Combination 31 X X X Combination 32 X X X Combination 33 X X X Combination 34 X X X Combination 35 X X X Combination 36 X X X Combination 37 X X X Combination 38 X X X Combination 39 X X X Combination 40 X X X Combination 41 X X X Combination 42 X X X Combination 43 X X X Combination 44 X X X Combination 45 X X X Combination 46 X X X Combination 47 X X X Combination 48 X X X Combination 49 X X X Combination 50 X X X Combination 51 X X X Combination 52 X X X Combination 53 X X X Combination 54 X X X Combination 55 X X X Combination 56 X X X Combination 57 X X X X Combination 58 X X X X Combination 59 X X X X Combination 60 X X X X Combination 61 X X X X Combination 62 X X X X Combination 63 X X X X Combination 64 X X X X Combination 65 X X X X Combination 66 X X X X Combination 67 X X X X Combination 68 X X X X Combination 69 X X X X Combination 70 X X X X Combination 71 X X X X Combination 72 X X X X Combination 73 X X X X Combination 74 X X X X Combination 75 X X X X Combination 76 X X X X Combination 77 X X X X Combination 78 X X X X Combination 79 X X X X Combination 80 X X X X Combination 81 X X X X Combination 82 X X X X Combination 83 X X X X Combination 84 X X X X Combination 85 X X X X Combination 86 X X X X Combination 87 X X X X Combination 88 X X X X Combination 89 X X X X Combination 90 X X X X Combination 91 X X X X Combination 92 Combination 93 X X X X X Combination 94 X X X X X Combination 95 X X X X X Combination 96 X X X X X Combination 97 X X X X X Combination 98 X X X X X Combination 99 X X X X X Combination 100 X X X X X Combination 101 X X X X X Combination 102 X X X X X Combination 103 X X X X X Combination 104 X X X X X Combination 105 X X X X X Combination 106 X X X X X Combination 107 X X X X X Combination 108 X X X X X Combination 109 X X X X X Combination 110 X X X X X Combination 111 X X X X X Combination 112 X X X X X Combination 113 X X X X X

Compositions

One aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 1 and further comprising a second compound selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. In one specific embodiment of the invention, the second compound may be Compound 2, Compound 3, Compound 4, Compound 5 or Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 2 and further comprising a second compound selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. In one specie embodiment of the invention, the second compound may be Compound 4. In one specific embodiment of the invention, the second compound may be Compound 3. In one specific embodiment of the invention, the second compound may be Compound 5.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 3 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. In one specific embodiment of the invention, the second compound may be Compound 1. In one specific embodiment of the invention, the second compound may be Compound 4. In one specific embodiment of the invention, the second compound may be Compound 5. In one specific embodiment of the invention, the second compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising a first compound selected from the group consisting of Compound 4 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. In one specific embodiment of the invention, the second compound may be Compound 1 or Compound 2 or Compound 3 or Compound 6. In one specific embodiment of the invention, the second compound may be Compound 1. In one specific embodiment of the invention, the second compound may be Compound 2. In one specific embodiment of the invention, the second compound may be Compound 3. In one specific embodiment of the invention, the second compound may be Compound 5. In one specific embodiment of the invention, the second compound may be Compound 8.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 5 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 8, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. In one specific embodiment of the invention, the second compound may be Compound 1. In one specific embodiment of the invention, the second compound may be Compound 8.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 6 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. In one specific embodiment of the invention, the second compound may be Compound 1. In one specific embodiment of the invention, the second compound may be Compound 2. In one specific embodiment of the invention, the second compound may be Compound 3. In one specific embodiment of the invention, the second compound may be Compound 4.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 6 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising a first compound selected from the group consisting of Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 18 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 1 and further comprising a second compound and a third compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 3, or Compound 4, or Compound 5 or Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 3. The second compound may be Compound 2 and the third compound may be Compound 5. The second compound may be Compound 3 and the third compound may be Compound 5.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 2 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15and Compound 18. The second compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may foe Compound 3 and the third compound may be Compound 5. The second compound may be Compound 3 and the third compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 3 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15and Compound 16. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 5.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising a first compound selected from the group consisting of Compound 4 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6. The second compound may be Compound 1 and the third compound may be Compound 5. The second compound may be Compound 2 and the third compound may be Compound 5.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 5 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 6 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3or Compound 4. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may foe Compound 1 and the third compound may be Compound 3. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 5. The second compound may be Compound 2 and the third compound may be Compound 5. The second compound may be Compound 3 and the third compound may be Compound 5.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 7 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15and Compound 16.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising a first compound selected from the group consisting of Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 1 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 3, Compound 4, Compound 5, or Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2, the third compound may be Compound 4, and the fourth compound may be Compound 6. The second compound may be Compound 3, the third compound may be Compound 4, and the fourth compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 2 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 3 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 4, the third compound may be Compound 5, and the fourth compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising a first compound selected from the group consisting of Compound 4 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 6. The second compound may be Compound 1 end the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 5 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 6 and further composing a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 15. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 4. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 7 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising a first compound selected from the group consisting of Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a composition, e.g., a pharmaceutical composition, the composition comprising Compound 1 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound

3, Compound 4, Compound 5 or Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 2 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6, The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 3 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 18. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising a first compound selected from the group consisting of Compound 4 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 5 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

The second compound may be Compound 1.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 6 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3, and Compound 4. The second compound may be Compound 1end the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising Compound 7 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 18.

Another aspect of the present invention includes a composition, e.g. a pharmaceutical composition, the composition comprising a first compound selected from the group consisting of Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Salts

The Combination Compounds and ether active ingredients can be in the form of a salt. Typically, but not absolutely, the salts of the Combination Compounds and other active ingredients are pharmaceutical acceptable sate. Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the Combination Compounds and/or other active ingredients. Examples of suitable pharmaceutically acceptable salts include inorganic acid addition salts such as chloride, bromide, sulfate, phosphate, and nitrate; organic acid addition salts such as acetate, galactarate, propionate, succinate, lactate, glycolate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate, and ascorbate; salts with acidic amino acid such as aspartate and glutamate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; organic basic salts such as trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, and N,N′-dibenzylethylenediamine salt; and salts with basic amino acid such as lysine salt and arginine salt. The salts may be in some cases hydrates or ethanol solvates.

Pharmaceutical Formulations

The Combination Compounds and/or other active ingredients can be formulated with conventional carriers or excipients, which can be selected in accord with ordinary practice. Tablets typically contain excipients, glidants, fillers, binders and the like. Aqueous formulations can be prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. All formulations will optionally contain excipients such as those set forth in the Handbook of Pharmaceutical Excipients (1986), herein incorporated by reference in its entirety. Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like.

The pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10.

While it is possible for an active ingredient to be administered alone it may be preferable to present one or more active ingredients as pharmaceutical formulations. The formulations of the invention, both for veterinary and for human use, comprise at least one active ingredient, together with one or mere acceptable carriers and optionally other therapeutic ingredients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.

The formulations include these suitable for the administration routes set forth below. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally can be found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.), herein incorporated by reference in its entirety. Such methods include the step of bringing into association an active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations can be prepared by uniformly and intimately bringing into association one or more active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of an active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. An active ingredient may also be administered as a bolus, electuary or paste.

A tablet can made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally can be formulated so as to provide slow or controlled release of an active ingredient

For administration to the eye or other external tissues e.g., mouth and skin, the formulations can be preferably applied as a topical ointment or cream containing an active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc), preferably 0.2 to 16% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, an active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, an active ingredient may be formulated in a cream with an oil-in-water cream base.

If desired, the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having too or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of an active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulphoxids and related analogs.

The oily phase of the emulsions of Combination Compounds and/or other active ingredients may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulation of the invention include Tween® 60 (ICI Americas Inc.), Span 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high malting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Pharmaceutical formulations according to the present invention comprise one or more active together with one or more pharmaceutically acceptable earners or excipients and optionally other therapeutic agents. Pharmaceutical formulations containing active ingredients may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing an active ingredient in admixture with nontoxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium of sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystallins cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be unseated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monestearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsules where an active ingredient(s) is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as sot gelatin capsules wherein an active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxy/propyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending an active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol sweetening agents, such as those set forth herein, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide an active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-wafer emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial asters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned herein. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of earner material which may vary from about 5 to about 95% of the total compositions (weight:weight). The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of an active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for administration to the eye include eye drops wherein an active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for an active ingredient. An active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth include lozenges comprising an active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising an active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising an active ingredient in a suitable liquid carrier. Formulations for rectal administration may be presented as a suppository with a suitable

comprising for example cocoa butter or a salicylate.

Formulations suitable for intrapulmonary or nasal administration have a particle size example in the range of 0.1 to 500 μm (including particle sizes in a range between 0.1 and

μm in increments such as 0.5 μm, 1 μm, 30 μm, 35 μm, etc.), which is administered by ra

inhalation through the nasal passage or by inhalation through the mouth so as to reach th

alveolar sacs. Suitable formulations include aqueous or oily solutions of an active ingredient. Formulations suitable for aerosol or dry powder administration may fee prepared according to conventional methods and may be delivered with other therapeutic agents such as compo

heretofore used in the treatment or prophylaxis of infections as described herein.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, paste, foams or spray formulations containing in addition to an ad

ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutions which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agent. The formulations can be presented in unit-dose or multi-dose containers, for example seal ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring the addition of the sterile liquid carrier, for example water for injection, immediately prior to

Extemporaneous injection solutions and suspensions can be prepared from sterile powder granules and tablets of the kind previously described. Preferred unit dosage formulations be those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned ab

the formulations of Combination Compounds and/or other active ingredients may include c

agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

Combination Compounds and other active ingredients can also be formulated to p

controlled release of an active ingredient to allow less frequent dosing or to improve the pharmacokinetic or toxicity profile of an active ingredient. Accordingly, the invention also provides compositions comprising two or more of the Combination Compounds formulated sustained or controlled release.

Dosages

The effective dose of an active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against an active disease or condition, the method of delivery, and the pharmaceutical formulation, and can be determined by the clinician using conventional dose escalation studies.

By way of example, compositions of the invention (e.g. tablets) can be formulated to provide effective doses. For example, with respect to Compound 1, or a pharmaceutically acceptable salt thereof, the composition may comprise from 1.0 mg to 100 mg, from 5 mg to 40 mg, from 30 mg to 50 mg, or 20 mg or 40 mg and can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 2, Compound 3, Compound 6, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. With respect to Compound 2 or a pharmaceutically acceptable salt thereof, the composition may comprise from 25 mg to 800 mg, from 50 mg to 400 mg, or from 60 mg to 300 mg or from 70 mg to 200 mg or may be 150 mg and can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 1, Compound 3, Compound 6, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. With respect to Compound 3, or a pharmaceutically acceptable salt thereof, the composition may comprise from 10 mg to 1000 mg, or 50 to 400 mg, or 100 mg to 400 mg or 200 mg to 400 mg and can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or mere of Compound 1, Compound 2, Compound 6, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. With respect to Compound 4, or a pharmaceutically acceptable salt thereof, the composition may comprise from 25 mg to 400 mg or from 25 mg to 200 mg can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 1, Compound 2, Compound 3, Compound 6, Compound 5 and Compound 7. With respect to Compound 5, or a pharmaceutically acceptable salt thereof, the composition may comprise from 50 mg to 1000 mg or 100 mg to 750 mg can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 1, Compound 2, Compound 3, Compound 6, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. With respect to Compound 6, or a pharmaceutically acceptable salt thereof, the composition may comprise from 1 mg to 500 mg or from 3 mg to 300 mg or from 3 mg to 200 mg or from 3 mg to 100 mg or from 10 mg to 90 mg or from 30 mg to 90 mg can be adapted to be administered one or mom times daily to a human being in need thereof in combination with any one or more of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. With respect to Compound 7, or a pharmaceutically acceptable salt thereof, the composition may comprise from 100 micrograms up to 3000 mg, from 25 mg up to 2000 mg, or from 50 mg up to 1000 mg and can be adapted to be administered one or more times daily (e.g. four times daily) to a human being in need thereof in combination with any one or more of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. With respect to Compounds 9 and 10, or pharmaceutically acceptable salts thereof, the composition may comprise 10 mg to 1000 mg per day (according to US 2010/0298257). With respect to Compound 11, or pharmaceutically acceptable salts thereof, the composition may comprise 1 mg to 1000 mg per day (according to US 2010/0081828). Dosages for Compounds 1-7 that are co-administered may need to be adjusted to account for potential drug-drug interactions. For example, although it does not appear that Compound 1 affects drug metabolizing systems, Compound 2 appears to have the effect of increasing the exposure of Compound 1approximately 2-3×. Therefore, a dose reduction (e.g. 2×-3×) of Compound 1 would be anticipated when Compound 1 is combined with Compound 2. In combination with Compound 16, Compound 2 appears to have the effect of increasing the exposure of Compound 6 approximately 5×, so dose reduction (e.g. 3×-5×) of Compound 16 would be anticipated when Compound 16 is dosed with Compound 2. Therefore, a 10 mg dose of Compound 6 when coadministered with Compound 2 approximate to a 30 mg dose.

The two or more Combination Compounds may be administered in conjunction with Ribavirin in amounts of about 800 mg, 1000 mg or 1200 mg per day in single or multiple dosages (e.g. about 400 mg, 500 mg or 600 mg twice daily).

Use of Combinations of the Invention

In practice of this aspect of the invention, Combination Compounds may be used in the dosages set forth above.

One aspect of the present invention includes Compound 1 for use in a method of treating HCV infections, wherein compound 1 is used in combination with a second compound selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 3, Compound 4, Compound 5 or Compound 6.

Another aspect of the present invention includes Compound 2 for use in a method of treating HCV infections, wherein compound 2 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4.

Another aspect of the present invention includes Compound 3 for use in a method of treating HCV infections, wherein compound 3 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6.

Another aspect of the present invention includes Compound 4 for use in a method of treating HCV infections, wherein Compound 4 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1 or Compound 2or Compound 3 or Compound 6.

Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infections, wherein Compound 5 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infections, wherein Compound 6 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4.

Another aspect of the present invention includes Compound 7 for use in a method of treating HCV infections, wherein Compound 7 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes Compound 9 for use in a method of treating HCV infections, wherein Compound 9 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7.

Another aspect of the present invention includes Compound 10 for use in a method of treating HCV infections, wherein Compound 10 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7.

Another aspect of the present invention includes Compound 11 for use in a method of testing HCV infections, wherein Compound 11 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7.

Another aspect of the present invention includes Compound 12 for use in a method of treating HCV infections, wherein Compound 12 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7.

Another aspect of the present invention includes Compound 13 for use in a method of treating HCV infections, wherein Compound 13 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7.

Another aspect of the present invention includes Compound 14 for use in a method of treating HCV infections, wherein Compound 14 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7.

Another aspect of the present invention includes Compound 15 for use in a method of treating HCV infections, wherein Compound 15 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7.

Another aspect of the present invention includes Compound 18 for use in a method of treating HCV infections, wherein Compound 16 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7.

Another aspect of the present invention includes Compound 1 for use in a method of treating HCV infections, wherein compound 1 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 3, or Compound 4, or Compound 5 or Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 2 for use in a method of treating HCV infections, wherein compound 2 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4. The second compound may be Compound 1and the third compound maw be Compound 4. The second compound may be Compound 1and the third compound may be Compound 6. The second compound may be Compound 4and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 3 for use in a method of treating HCV infections, wherein compound 3 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 4 for use in a method of treating HCV infections, wherein Compound 4 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infections, wherein Compound 5 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infections, wherein Compound 6 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11 Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4.

Another aspect of the present invention includes Compound 7 for use in a method of treating HCV infections, wherein Compound 7 is used in combination with a second compound end a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes Compound 9 for use in a method of treating HCV infections, wherein Compound 9 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 8 and Compound 7.

Another aspect of the present invention includes Compound 10 for use in a method of treating HCV infections, wherein Compound 10 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes Compound 11 for use in a method of treating HCV infections, wherein Compound 11 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes Compound 1 for use in a method of treating HCV infections, wherein compound 1 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 3, Compound 4, Compound 5, or Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 2 for use in a method of treating HCV infections, wherein compound 2 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 6. Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 3 for use in a method of treating HCV infections, wherein compound 3 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 4 for use in a method of treating HCV infections, wherein Compound 4 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infections, wherein Compound 5 is used in combination with a second compound, e third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infections, wherein Compound 6 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 4. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4.

Another aspect of the present invention includes Compound 7 for use in a method of treating HCV infections, wherein Compound 7 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes Compound 9 for use in a method of treating HCV infections, wherein Compound 9 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes Compound 10 for use in a method of treating HCV infections, wherein Compound 10 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes Compound 11 for use in a method of treating HCV infections, wherein Compound 11 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes Compound 1 for use in a method of treating HCV infections, wherein compound 1 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 3, Compound 4, Compound 5 or Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 2 for use in a method of treating HCV infections, wherein compound 2 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 3 for use in a method of treating HCV infections, wherein compound 3 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 4 for use in a method of treating HCV infections, wherein Compound 4 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6.

Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infections, wherein Compound 5 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infections, wherein Compound 6 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2. Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 4. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4.

Another aspect of the present invention includes Compound 7 for use in a method of treating HCV infections, wherein Compound 7 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes Compound 9 for use in a method of treating HCV infections, wherein Compound 9 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes Compound 10 for use in a method of treating HCV infections, wherein Compound 10 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes Compound 11 for use in a method of treating HCV infections, wherein Compound 11 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

One aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method composing administering Compound 1 and further comprising administering a second compound selected from the group consisting of comprising Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 3, Compound 4, Compound 5 or Compound 6.

Another aspect of the present invention includes a method for ameliorating one or symptom of HCV infection in a human, a method for reducing viral load in a burners diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 2 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral lead in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 3 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 4Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagno

with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 4 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2 or Compound 3 or Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 5 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 6 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 7 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 9 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 10 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 11 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the presort invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 1 and further composing administering a second compound and a third compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15and Compound 16. The second compound may be Compound 3, or Compound 4, or Compound 5 or Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 2 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 3 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may at Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministerad oral antiviral agents, each method comprising administering Compound 4 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 5 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 6 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 1S and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 7 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 9 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 10 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 11 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 1 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 18. The second compound may be Compound 3, Compound

Compound 5, or Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagr

with HCV, a method of treating HCV in a human subject, and a method for reducing emerg

of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 2 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consist of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or n

symptom of HCV infection in a human, a method for reducing viral load in a human diagnos

with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 3 and further composing administering a second compound, a third compound and a fourth compound each selected from the group consist of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound

The second compound may be Compound 1 and the third compound may be Compound 5. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 4 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consists of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 6 second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6.

Another aspect of the present invention includes s method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral lead in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 5 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consis

of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 8 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 4. The second compound may be Compound 1 and third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 4 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 4. The second compound may be Compound 3 and the third compound may be Compound 4.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 7 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 9 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagno; with HCV, a method of treating HCV in a human subject, and a method for reducing emerg of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 10 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consist of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 11 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consist of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 1 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 3, Compound 4, Compound 5 or Compound 6. The second compound may be Compound and the third compound may be Compound 4. The second compound may be Compound

and the third compound may be Compound 4. The second compound may be Compound

and the third compound may be Compound 6. The second compound may be Compound

and the third compound may be Compound 6. The second compound may be Compound

and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 2 and former comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound

. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 3 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound

or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 4 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 4 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7. The second compound may be Compound 1, Compound 2″, Compound 3 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 2. The second compound may be Compound 1 and the third compound may be Compound 3. The second compound may be Compound 1 and the third compound may be Compound 6. The second compound may be Compound 2 and the third compound may be Compound 6. The second compound may be Compound 3 and the third compound may be Compound 6.

Another aspect of the present invention includes a method for ameliorating one or symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treeing HCV in a human subject, and a method for reducing emer

of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 5 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected for group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosing with HCV, a method of treating HCV in a human subject, and a method for reducing emerg

of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 8 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3, and Compound 4. The second compound may be Compound

and the third compound may be Compound 2. The second compound may be Compound

and the third compound may be Compound 3. The second compound may be Compound

and the third compound may be Compound 6. The second compound may be Compound

and the third compound may be Compound 4. The second compound may be Compound

and the third compound may be Compound 4.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method composing administering Compound 7 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject and a method for reducing emergence of HCV quaslspecles with resistance to coadministered oral antiviral agents, each method comprising administering Compound and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 10 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, Compound 7.

Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 11 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 3Compound 7.

Two or more of Compound 1, Compound 2, Compound 3, Compound 4, Compound

, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and any other componen

a combination therapy can be adapted to be administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and the like. It will be appreciated that the primed r

may vary with, for example, the condition of the recipient.

A synergistic effect may be attained when the active ingredients are: (1) co-formula (e.g. in a unitary dosage form) and administered or delivered simultaneously in a combines formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by so other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., in separate tablets, or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e. ser

whereas in combination therapy, of effective dosages of two or more active ingredients are administered together.

Co-administration of a Combination Compound with one or more Combination Compounds generally refers to simultaneous or sequential administration of one or more Combination Compounds, such that therapeutically effective amounts of two or more Combination Compounds are present in the body of the patient. In some cases, Combination Compounds (e.g. two, three or four Combinations Compounds) will be co-formulated to a

administration at the same time, in some cases, co-formulated Combination Compounds i

be co-administered with one or more additional Combination Compounds.

Co-administration also includes administration of unit dosages of the Combination Compounds before or after administration of unit dosages of one or more other active ingredients, for example, administration of two or more Combination Compounds within seconds, minutes, or hours of the administration of one or more other active ingredients. For example, a unit dose of a Combination Compound can be administered first, followed within seconds or minutes by administration of a unit dose of a second Combination Compound, followed within seconds or minutes by administration of a unit dose of one or more other as Ingredients. Alternatively, a unit dose of one or more other active ingredients can be administered first, followed within seconds or minutes by administration of a unit dose of a Combination Compound, followed within seconds or minutes by administration of a unit dose a second Combination Compound. In some cases, if may be desirable to administer a unit dose of a Combination Compound first, followed, after a period of hours (e.g., 1-12 hours), administration of a unit dose of a second Combination Compound, followed, after a period hours (e.g., 1-12 hours), by administration of a unit dose of one or more other active ingredients. In other cases, it may be desirable to administer a unit dose of one or more of active ingredients first, followed, after a period of hours (e.g., 1-12 hours), by administrator unit dose of a Combination Compound, followed, after a period of hours (e.g., 1-12 hours), administration of a unit dose of a second Combination Compound. Where three or more Combinations Compounds are administered with one or more additional active ingredients, Combination Compounds may be administered one after another within seconds, minutes, hours (e.g. 1-12 hours) of each other and the one or more additional active ingredients may be administered before, during or after the administration of the Combination Compounds. W

Combination Compounds are co-formulated, they can be administered simultaneously, or before or after the administration of one or mom additional active ingredients.

Unless otherwise specified, the combination therapy may be administered as sepa

dosage forms with each active ingredient, administered together or separately, sequentially concurrently, and dose in time or remote in time to each other.

The course of treatment can extend, for example, from about 12 weeks to about 48 weeks, or longer, for example, from about 12 weeks to about 24 weeks.

The present invention includes a combination of therapeutically effective component ameliorate at least one symptom of HCV infection in a human being including, but not limit nausea, vomiting, loss of appetite, fatigue, jaundice, vomiting, diarrhea, dehydration, abdominal pain, cirrhosis of the liver. In addition, in some HCV infected individuals the use of combination therapy is effective to reduce the viral load of HCV viral particles present in the body of the infected person by a statistically significant amount. Viral load can be measured, for example, by measuring plasma HCV RNA levels using, for example, the COBAS TtqMan HCV ass

(Roche Molecular Systems). Typically, an HCV infected person who is treated with the Combination Compounds in accordance with the present invention experiences an improvement in one or all of the symptoms associated with the HCV infection.

Combinations of Two or More of the Combination Compounds with Ribavirin but not Interferon

As discussed above, some current HCV treatments include the administration of interferon, but this treatment typically produces unwanted side effects. Therefore it would desirable to find effective HCV treatments that do not require the administration interferon.

One aspect of the present invention provides for compositions, methods, uses and like for the treatment of HCV comprising administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without administering one or more interferons. This aspect of the invention may be particularly useful because it allows for the effective treatment of HCV without the side effects associated with the administration of one or more interferon.

In one embodiment of the present invention, the combined amount of ribavirin and Combination Compounds or pharmaceutically acceptable salts thereof, optionally with one more additional agents, is effective to treat HCV infection.

Another aspect of the present invention includes a method for ameliorating one or more symptoms of HCV infection in a human comprising: administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without concurrent administration of one or more interferon. In this regard, the present invention d

not foreclose the potential for dosing one or mere interferon. Rather, the present invention be used in conjunction with another therapy that, in fact, includes one or more interferon. An aspect of the present invention includes efficacious treatment of HCV with ribavirin without need for one or more interferon.

Another aspect of the present invention includes a method for reducing viral toed in a human diagnosed with HCV comprising; administering two or more of the Combination Compounds or pharmaceutically acceptable sets thereof and ribavirin, but not one or more interferon.

Another aspect of the present invention includes a method for treating HCV in a human subject consisting essentially of administration of ribavirin in conjunction with two or more of the Combination Compounds or pharmaceutically acceptable salts thereof.

Another aspect of the present invention includes a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents comprising: administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without concurrent administration of one or more interferon.

Similarly, another aspect of the present invention includes a composition, e.g. a pharmaceutical composition for ameliorating one or more symptom of HCV infection in a human comprising two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without one or more interferon. Another aspect of the present invention includes a composition for reducing viral load in a human diagnosed with HCV comprising two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, but not one or more interferon. Another aspect of the present invention includes a composition for treating HCV in a human subject consisting essentially of ribavirin in conjunction with two or more of the Combination Compounds or pharmaceutically acceptable salts thereof. Another aspect of the present invention includes a composition for ribavirin-based HCV therapy comprising two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, with the proviso that said composition does not include one or more interferon. Another aspect of the present invention includes a composition for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents comprising two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without one or more interferon.

Similarly, another aspect of the present invention includes use of: two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without one or more interferon, in the manufacture of a medicament for ameliorating one or mom symptoms of HCV infection in a human; as well as use of: two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, but not one or more interferon, in the manufacture of medicament for reducing viral load in a human diagnosed with HCV; as well as use of ribavirin in conjunction with two or more of the Combination Compounds or pharmaceutically acceptable salts thereof in the manufacture of a medicament for treating HCV in a human subject, wherein said use does not include use of one or more interferon; as well as use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, in the manufacture of a medicament for ribavirin-based HCV therapy, wherein said use avoids administration of one or more interferon; as well as use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without one or more interferon in the manufacture of a medicament for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents.

Another aspect of the present invention includes a combination comprising ribavirin and two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, which combination is substantially free of one or more interferon. In one embodiment, the combination may occur as separate dosage forms with each active ingredient, administered together or separate, sequentially or concurrently, and close in time or remote in time to each other.

Another aspect of the present invention includes a kit comprising: ribavirin, two or more of the Combination Compounds and instruction regarding a treatment regimen to treat, reduce viral load, or delay onset or progression of HCV wherein the treatment regimen includes administration of the two or more of the Combination Compounds and ribavirin without administration of one or more interferon. In one embodiment, such a lot may also include packaging, such as a blister pack. Alternatively, such a kit may provide for individual prescription and dosing of each component as separately packaged pharmaceutics, but when combined with the instruction regarding a treatment regimen to treat, reduce viral load, or delay onset or progression of HCV, such is intended to be within the scope of the present invention.

Another aspect of the present invention includes a pharmaceutical composition comprising: ribavirin; two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers. In one embodiment, the pharmaceutical composition may be a unitary dosage form.

Unless otherwise specified, the combination therapy with Ribavirin may be administered as separate dosage forms with each active ingredient administered (including the Combination Compounds), may be administered together (e.g., in the form of a unit dosage, such as a tablet) or separately, sequentially or concurrently, and close in time or remote in time to each other, if administered separately, each compound may be administered with the other(s) at the same time, or either before or after such administration of the other(s). The active ingredients can be administered daily. In one embodiment, a daily dosage of the active ingredients is administered in separate sub-doses, such as one, two, three or four times per day. Advantageously, the daily dosage of Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin may be administered once per day.

Although the present invention includes compositions, methods, uses and the like for the treatment of HCV comprising administering two or more Combination Compounds or a pharmaceutically acceptable salt thereof; and ribavirin, but not one or more interferon, the present invention does not foreclose the potential for dosing one or more interferon to the human. Rather, the present invention may be used in conjunction with another therapy for another indication that, in fact, includes one or more interferon.

Combinations of Two or More Compounds with Ribavirin and Interferon

Another aspect of the present invention provides for compositions, methods, uses and the like comprising administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, and one or more interferon for treatment of HCV. The administration of more interferon may be in temporal relation to the administration of the Combination Compounds and ribavirin.

Another aspect of the present invention includes a method for ameliorating one or more symptoms of HCV infection in a human comprising administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, ribavirin, and one or more interferons. Another aspect of the present invention includes a method for reducing viral load in a human diagnosed with HCV comprising: administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof along with ribavirin and one or more interferons.

Another aspect of the present invention includes a method of ribavirin-based HCV therapy comprising administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof along with ribavirin, and one or more interferons.

Another aspect of the present invention includes a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents comprising: administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof along with ribavirin and one or more interferons.

Another aspect of the present invention includes use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof ribavirin, and one or more interferons, in the manufacture of a medicament for ameliorating one or more symptoms of HCV infection in a human. Another aspect of the present invention includes use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof along with ribavirin and one or more interferons, in the manufacture of medicament for reducing viral load in a human diagnosed with HCV. Another aspect of the present invention includes use of ribavirin in conjunction with two or more of the Combination Compounds or pharmaceutically acceptable salts thereof in the manufacture of a medicament for treating HCV in a human subject, wherein said use includes use of one or more interferons. Another aspect of the present invention includes use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, in the manufacture of a medicament for ribavirin-based HCV therapy, wherein said use includes administration of one or more interferon. Another aspect of the present invention includes use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, ribavirin, and one or more interferons in the manufacture of a medicament for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents.

Another aspect of the present invention includes a combination comprising ribavirin and two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, which combination includes one or more interferons.

Another aspect of the present invention includes a kit comprising: ribavirin, two or more of the Combination Compounds and one or more interferon; and instructions regarding a treatment regimen to treat, reduce viral load, or delay onset or progression of HCV wherein the treatment regimen includes administration of the two or more of the Combination Compounds and ribavirin and administration of one or more interferon. In one embodiment, such a kit may also include packaging, such as a blister pack. Alternatively, such a kit may provide for individual prescription and dosing of each component as separately packaged pharmaceutics, but when combined with the instruction regarding a treatment regimen to treat, reduce viral load, or delay onset or progression of HCV, such is intended to be within the scope of the present invention.

Another aspect of the present invention includes a pharmaceutical composition comprising: two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, ribavirin, and one or more interferon; and one or more pharmaceutically acceptable carriers. In one embodiment, the pharmaceutical composition may be a unitary dosage form.

Unless otherwise specified, the combination therapy with Ribavirin and one or more interferons may be administered as separate dosage forms with the one or more interferons administered to the patient and each of the remaining active ingredients to be employed in the combination therapy (including the Combination Compounds) are administered together (e.g., in the form of a unit dosage, such as a tablet) or separately, sequentially or concurrently, and close in time or remote in time to each other. If administered separately, each active ingredient may be administered with the other(s) at the same time, or either before or after such administration of the other(s). The active ingredients can be administered daily. In one embodiment, a daily dosage is administered in separate sub-doses, such as one, two, three or four times per day.

Combination Therapy, Including Additional Therapeutics

In another embodiment, non-limiting examples of suitable combinations include the combinations of two or more of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 with one or more additional active ingredients including HCV NS3 protease inhibitors, alpha-glucosidase 1inhibitors, hepatoprotectants, nucleoside or nucleotide inhibitors of HCV NS5B polymerase, non-nucleoside inhibitors of HCV NS5B polymerase, HCV NS5A Inhibitors, TLR-7 agonists, cyclophilin inhibitors, HCV IRES inhibitors, HCV entry inhibitors, HCV maturation inhibitors, HCV assembly inhibitors, HCV infectivity inhibitors and pharmacokinetic enhancers, as well as other drugs for treating HCV. More specifically, one or more compounds of the present invention may be combined with one or more compounds selected from the group consisting of:

-   -   (i) HCV NS3 protease inhibitors, e.g., boceprevir (SCH-503034,         SCH-7), telapravir (VX-950), TMC-435 (IUPAC N-[(2R, 3aR,10Z,         11aS, 12aR,         14aR)-2-[2-(4-isopropylthiazol-2-yl)-7-methoxy-8-methhyiquinolin4-yloxy]-5-methyl-4,14-dioxo-1,2,3,3a,4,5,6,7,8,9,11a,12,12a,13,14,14a-hexadecahydrocyclopenta[c]cylclopropa[g]H[1,6]diazacyclotetradecin-12a-ylcarbony]cyclopropanesulfonamide],         ABT-450, ACH-1625, ACH-2684, BI-201335, BI-1230, MK-5172,         MK-7009, SCH-900518, VBY-376, VX-500, GS-9256, GS-9451,         BMS-605339, PHX-1766, AS-101, YH-5258, YH-5530, YH-5531, and         ITMN-191 (R-7227);     -   (ii) alpha-glucosidase 1 inhibitors, e.g., celgosivir (MX-3253),         UT-231B, Miglitol;     -   (iii) hepatoprotectants, e.g., emericasan (IDN-6556), ME-3738,         silibilin, and MitoQ;     -   (iv) nucleoside or nucleotide inhibitors of HCV NS5B polymerase,         e.g., R1626, R7128 (R4048), IDX184, IDX-102, PSI-661, PSI-938,         PSI-7851, PSI-7977, BCX-4878, valopicitabine (NM-283), MK0608         and TMC649128;     -   (v) non-nucleoside inhibitors of HCV NS5B polymerase, e.g.,         filibuvir (PF-868554), ABT-333, ABT-072, BI-207127, VCH-759,         VCH-916, JTK-652, MK-3281, VBY-708, VCH-222, A848837, ANA-598,         GL60667, GL59728, A-63890, A-48773, A-48547, BC-2329, VCH-796         (nesbuvir), GSK625433, BILN-1941, and XTL-2125;     -   (vi) HCV NS5A inhibitors, e.g., ACH-2928, AZD-2836 (A-831),         AZD-7295 (A-689), BMS-766, BMS-790052, BMS-824393, and PPI-461;     -   vii) TLR-7 agonists, e.g., imiquimod, 852A, ANA-773, ANA-975,         AZD-8848 (DSP-3025), PF-04878691, and SM-360320 and Compound 8;     -   (viii) cyclophilin inhibitors, e.g., DEBIO-025, SCY-635, and         NIM811;     -   (ix) HCV IRES inhibitors, e.g., MCI-067;     -   (x) pharmacokinetic enhancers, e.g. roxythromycin, BAS-100,         SPI-452, PF-4194477, TMC-41629;     -   (xi) HCV entry inhibitors;     -   (xii) HCV assembly inhibitors;     -   (xiii) HCV maturation inhibitors;     -   (xiv) HCV infectivity inhibitors; and     -   (xv) other drugs for treating HCV, e.g., thymosin alpha 1         (Zadaxin), nitazoxanide (Alinea, NTZ), BIVN-401 (virostat),         PYN-17 (altirex), KPE02003002, actilon (CPG-10101), KRM-7000,         civacir, GI-5005, XTL-6885, BIT225, PTX-111 ITX2865, TT-033i,         ANA 971, NOV-205, tarvacin, EHC-18, VGX-410C, E         702, AVI 4065, BMS-650032, BMS-791325, Bavtuximab, MDX-1106 (O         4538), Oglufanide, FK-788, and VX-497 (merimepodib).

Synthetic Examples

Synthetic protocols for the preparation of Compounds 1, 2, 3, 6, 7, and 8 are knows the literature. Additionally, a synthetic protocol for preparing each of the combination Compounds is provided in the Examples below.

Compound 1 can be prepared using synthetic methods and intermediates like those described so U.S. Pat. No. 7,754,720. Compound 1 can also be prepared as described in the following Example.

Example 1 5-((6-[2,4-bis(trifluoromethyl)phenyl]pyridazin-3-yl)methyl)-2-(2-fluorophenylimidazo[4,5-c]pyridine 1

1 Compound MW Amount Moles Equivalents 104  453.79 95 mg 0.209 1 DME  500 μL 2N aq. Na₂CO₃ 313 μL 0.626 3 105  257.93 80.9 mg 0.313 1.5 Pd(PPh₃)₄ 1155 12 mg 0.0104 0.05

Compound was dissolved in dimethoxyethane (DME). To this solution was ad

2,4-(trifluoromethyl)phenylboronic acid 105 and a 2N aq. Na₂CO₂ solution. To the resulting biphasic mixture was added Pd(PPh₃)4 and the reaction was then heated at 80° C. for 72 hrs. The reaction was cooled to room temperature and filtered through Celite and the Celite was with EtOAc. The filtrate was concentrated in vacuo. The residue was purified on 6 g SiO₂ u

MeOH/CH₂Cl₂ to elute compound. The compound thus obtained was contaminated with PPh₃(O). The product was repurified on a 1 mm Chromatotron plate with 0 to 5% MeOH/CH₂Cl₂ in 1% steps. The pure fractions were combined and concentrated its vacuo, dried on high vacuum for 12 hrs. 11.8 mg of the free base of compound 1 was obtained with PPh_(s) contamination. ¹H NMR (300 MHz, CD₃OD) δ 6.20 (s, 2), 7.32 (m, 1), 7.52 (m, 1), 7.7, 1), 7.89 (d, 1), 7.95 (s, 2), 8.15 (m, 3), 8.35 (d, 1), 9.12 (s, 1); LC/MS M+H=518.

The intermediate compound 104 was prepared as follows

a. Preparation of Compound 102.

Compound MW Amount mmoles Equivalents 101 128.56 5 g 38.9 1 TCCA 232.41 3.62 g 15.6 0.4 CHCl₃ 130 mL

To a solution of the commercially available starting material 101 in CHCl₃, trichloroisocyanuric acid (TCCA) was added at 60° C. Then the solution was stirred for 1.5 cooled, and filtered with HiFlo-Celite. The filtrate was concentrated and dried with vacuum. yield was 5.037 g of compound 102.

b. Preparation of Compound 104.

Compound MW Amount mmoles Equivalents 102 163 5.073 g 31.12 1 103 213.2 6.635 g 31.12 1 NaOH (10%) 40 1.245 g 31.12 1 DMF 320 mL

To a solution of compound 183 in DMF (dimethylformamide), NaOH was added. Compound 102 was dissolved in DMF (20 mL) and added to the solution slowly. The reaction was stirred for 3 hrs, was diluted with water and extracted with EtOAc. The organic layer

dried with Na₂SO₄. The solvent was mmoved and the product recrystallized with dichloromethane. The yield was 5.7 g of compound 103.

Compound 2 can be prepared using synthetic methods and intermediates like those described in U.S. Ser. No. 12/202319 (US 20100051783 A1), Compound 2 can also be prepared described in the following Example.

Example 2 Preparation of Compound 2

Phosphinate ester 206 (23.7 g, 24.05 mmol) was dissolved in CH₃CN (240 mL) and cooled to 0° C. Iodotrimethylsilane (17.4 mL, 122.3 mmol) was added at a fast drop-wise p

followed by, after 10 mm, 2,6-lutidine (17.0 mL, 146.4 mmol). The reaction mixture was sl

warmed to room temperature and stirred for 1 h then cooled back down to 0° C. and 2,6-lutidine (11.1 ml, 95.6 mmol) followed by MeOH (24 mL) were added. The solution was concentration in vacuo and the crude residue was purified by HPLC to afford 12.68 g of Compound 2 in

yield. ¹H NMR (300 MHz, CDCl₃) δ 8.35 (d, J=9.3 Hz, 1H), 8.28 (s, 1H), 7.65 (s, 1H), 7.6 J=9.6 Hz, 1H), 7.35-7.22 (m, 1H), 7.02-6.89 (m, 2H), 5.85 (bs, 1H), 4.82-4.71 (m, 2H), 4.3 (bs, 1H), 4.28-3.99 (m, 3H), 4.16 (s, 3H), 3.57-3.28 (m, 2H), 2.90-2.78m, 1H), 2.63-2.50 (m, 1H), 2.08-1.91 (m, 1H), 1.91-170 (m, 2H), 1.70-1.13 (m, 22H), 1.37 (d, J=6.9 Hz, 6H); 31R NMR (121.4 MHz, CD₃OD) δ42.4; LCMS (M+1); 957.35. g.

Intermediate compound 206 was prepared as follows.

a. Preparation of Compound 203.

Compound 201 (17.42 g, 28.30 mmol) was dissolved in THF (138 mL) and cooled

0°C. To the solution was added N-methylmorpholine (4.7 mL, 42.7 mmol). After 10 min at i-butylchloroformate (4.05 mL, 30.96 mmol) was added dropwise. After an additional 1 h, ( amino-2-vinyl-cyclopropyl)-(2,6-difluoro-benzyl)-phosphinic acid ethyl ester (8.94 g, 29

mmol) was slowly added as a solution in THF (20 mL). The suspension was warmed to room temperature and after 2 h it was partitioned between H2O (400 mL) and ethylacetate (200

. The aqueous layer was extracted with ethylacetate (200 ml×2) and the combined organic layers were washed with HCl (1N, 225 mL) and H2O (200 mL). The acid wash and aqueous wash were combined and back-extracted with ethylacetate (175 mL×2, 100 mL×2). The combined organic layers were washed with brine (400 mL), dried over Na2SO4, and concentrated in vacuo providing 25.06 g of diene 203 in 98.5% crude yield. LCMS (M+1) 898.06.

b. Preparation of Compound 204.

Compound 203 (12.91 g, 14.36 mmol) was dissolved in CH₂Cl₂ (1440 mL) and the solution was degassed for 30 minutes. The solution was heated 40° C. and Grubb's G1 catalyst (2.95 g, 3.59 mmol) was added. The reaction was refluxed for 17 h whereupon tris-hydroxymethylphosphine (22.3 g, 18.0 mmol), TEA (50 mL, 35.9 mmol), and H₂O (400 mL) were added and the reaction mixture was heated to reflux for an additional 10 hours. The reaction mixture was cooled to room temperature and the two layers were separated. The organic layer was washed with H₂O (400 mL) and brine (300 mL), dried over MgSO4, and concentrated. The crude residue was purified by silica-gel chromatography to afford 8.30 g macrocyclic olefin 204 in 66% yield. LCMS (M+1): 870.09.

c. Preparation of Compound 208.

The macrocyclic olefin 204 (7.34 g, 8.42 mmol) was dissolved in ethylacetate (105

and rhodium on alumina (5% wt, 2.945 g, 0.40 wt %) was added. The system was evacuated and flushed with H₂ (1 atm, 3×). To the system, after 3 h, was added more rhodium on alu

(5% wt, 842 mg, 0.10 wt %) and evacuated and flushed with H₂ (1 atm, 3×). After an additional 1 h the suspension was filtered and concentrated in vacuo providing 6.49 g of reduced macrocycle 205 in 88% crude yield. LCMS (M+1): 872.04.

d. Preparation of Compound 206.

The brosylate macrocycle 205 (6.49 g, 7.67 mmol) was dissolved in N-methylpyrrolidinone (25.0 mL) and 8-chloro-2-(2-isopropylamino-thiazol-4-yl)-7-methoxy-quinolin-4-ol 207 (2.564 g, 7.33 mmol) followed by Cs₂CO₃ (4.40 g, 13.50 mmol) were added. The mixture was heated to 65° C. for 8 h then diluted with ethylacetate (200 mL) and washed with LiCl (5%, 250 mL). The aqueous layer was extracted with ethylacetate (100 ml×2)

the combined organic layers were washed with brine (150 mL), dried over Na₂SO₄/MgSO₄, concentrated in vacuo. The crude residue was purified via silica-gel chromatography (ethylacetate-methanol) affording 4.39 g of aminothiazole 206 in 58% yield. LCMS (M+1) 985.28.

Intermediate Compound 201 can be prepared as follows.

d. Preparation of Compound 209 .

Compound 208 (7.00 g, 28.55 mmol) and DABCO (5.13 g , 45.94 mmol) wore dissolved in toluene (30 mL). A toluene (11 mL) solution of brosylchloride (10.22 g, 40.01 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction was diluted with EtOAc (210 mL) and 0.5N HCl (200 mL) was added. The two layers ware separated and the aqueous layer was extracted with EtOAc (2×200 mL). The combined organic layers were washed with brine (200 mL), dried with Na₂SO₄, filtered, and concentrated. The crude product was purified by combi-flash to give 12.23 g of compound 209 in 92% yield.

f. Preparation of Compounds 210 and 212.

Compound 2m (12.2 g, 26.3 mmol) was treated with 4 N HCl/1,4-dioxane (60 mL) and stirred for 1 hour. The reaction mixture was concentrated and dned under vacuum for 20 minutes. The crude amine HCl salt of compound 210 was dissolved in DMF (150 mL) and acid 211 (14.2 g, 52.6 mmol) was added. HATU (20.0 g, 52.6 mmol) and NMM (13.5 g, 131.5 mmol) were added. The reaction mixture was stirred at room temperature overnight. The reaction was diluted with EtOAc (300 mL), washed with 1 N HCl (200 mL), saturated NaHCO₃, brine, dried with Na₂SO₄, and concentrated. The crude product was purified by combi-flash to give 15.1 g of compound 212 in 93% yield,

g. Preparation of Compound 213.

To a solution of 212 (12.8 g, 20.7 mmol) in CH₂Cl₂ (50 mL) was added 4 N HCS in 1,4-dioxane (50 mL, 200 mmol). The reaction mixture was stirred at room temperature for 2 hours, concentrated, dried under vacuum for 20 minutes, and then dissolved in CH₃CN (50 mL). Saturated NaHCO₃ in H₂O (50 mL) was added and stirred for 5 minutes. Freshly prepared cyclopentylchloroformate in THF (50 mL) was added. The reaction was complete within 1 h, The solvent was removed under reduced pressure and the residue was diluted with EtOAc. The mixture was brought to pH=2 with 1 N HCS and the two layers were separated. The organic layers were washed with brine, dried with Na₂SO₄, filtered, and concentrated to give crude compound 213 (3.18 g),

h. Preparation of Compound 201.

The crude ester 213 (3.18 g, 5.07 mmol) was dissolved in THF (25 mL), H₂O (25 mL), and then MeOH (6 mL) and LiOH (660 mg, 25.4 mmol) was added. The reaction mixture was stirred at room temperature for 1 h and diluted with EtOAc. The reaction mixture was acidified to pH 2 with 1 N HCl and the two layers were separated. The aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed wits brine, dried with Na₂SO₄ concentrated and dried under vacuum to give 3.09 g of acid 201.

Intermediate 8-chloro-2-(2″isopropylamino-thiazol-4-yl)-7-methoxy-quinolin-4-ol 207 can be prepared as follows.

i. Preparation of 8-chloro-4-hydroxy-7-methoxyquinoline-2-carboxylic acid 215.

To a solution of methyl 8-chloro-4-hydroxy-7-methoxyquinoline-2-carboxylate 214 (36.5g, 0.145 mol) in a mixture of 1:1 of MeOH: THF (160 mL total) was added a solution of LiOH (30.5 g, 0.725 mol) in H₂O (80 mL). The mixture was stirred at room temperature for an hour when LCMS analysis showed complete conversion to the carboxylic acid. The reaction was worked up by removal of the volatiles and adjusting the pH of the solution to 6 using aqueous 6N HCl. The resulted gummy residue was filtered and dried on the lyophilizer for 2 days to provide 34.4 g (99.6%) of compound 215 as a white solid. EI MS (m/z) 253.9 [M+H].

j. Preparation of 2-(2-diazo-1-oxo)-8-chloro-7-methoxyquinolin-4-yl isobutyl carbonata 216.

To a solution of 8-chloro-4-hydroxy-7-methoxyquinoline-2-carboxylic acid 215 (10.2 g, 0.04 mol) in THF (400 mL) was added triethyl amine (12.3 ml, 0.088 mol) and i-Butylchloroformata (11.6 ml, 0.088 mol) at 0° C. under an argon atmosphere. The mixture was stirred at 0° C. for 1 hour when LCMS analysis demonstrated completion of the reaction to provide the desired mixed anhydride. EI MS (m/z) 454.0 [M+H]. To the reaction mixture of the anhydride was added a 1M solution of diazomethane (121 mL, 0.121 mol) in diethyl ether via a plastic funnel at 0° C. This mixture was allowed to stir while warming up to room temperature for additional 2 hours. Analysis of the mixture by LCMS demonstrated completion of the reaction. The septum was removed and the reaction was stirred for additional 20 minutes before removal of the solvent. The residue was dried further under high vacuum to provide compound 216, which was carried on to the next step. EI MS (m/z) 377.9 [M+H].

k. Preparation of 8-chloro-2-(2-(isopropylamino)thiazol-4-yl)-7-methoxyquinolin-4-ol 207.

To a cooled solution of 2-(2-diazo-1-oxo)-8-chloro-7-methoxyquinolin>4-yl isobutyl carbonate 216 (15.2 g, 0.040 mol) at 0° C. in THF (268 mL) was added 48% HBr (23 mL, 0.201 mol) slowly over 15 minutes. The solution was stirred at 0° C. for an additional 40 minutes when LCMS analysis demonstrated complete reaction. The reaction was worked up by addition of aqueous 1N NaOH (180 mL) at 0° C. to adjust the pH of the aqueous layer to 9. The layers were separated and the aqueous layer was washed with EtOAc (2×200 mL). Combined organic extracts were washed with brine and dried over MgSO4. The solvent was removed in vacuo to provide 17.7 g of a yellow solid. EI MS (m/z) 4³¹.9 [M+H].

The solution of the bromoketone obtained from the previous reaction was suspended in i-propanol (270 mL) and isopropylisourea (9.4 g, 0.080 mol). The reaction mixture was heated at 72° C. for 32 hours. LCMS analysis of the reaction demonstrated complete conversion to the desired-product. The reaction was allowed to cool to room temperature to allow for the product to precipitate out of the solution. The reaction was further cooled to 0° C. for 12 hours before filtration. The filtrate was washed with ether and dried on lyopholizer to provide 8.03 g of compound 207 as an orange solid. ¹H NMR (500 MHz, CDCl₃): δ 8.21 (d, J=9 Hz, 1H), 7.74 (s, 1H), 7.44 (d, J=10 Hz), 1H), 7.07 (s, 1H), 4.05 (s, 3H), 3.92 (penlet, J=6 Hz, 1H), 1.25 (d, J=7 Hz, 6H): EI MS (m/z) 350.0 [M+H].

Compound 3 can be prepared using synthetic methods and intermediates like those described in U.S. Ser. No. 12/215,808 (US 20090257978 A1). Compound 3 can also be prepared as described in the following Example.

Example 3 Preparation of Compound 3

Compound 318 (12 g, 13 mmol) was dissolved in THF (200 mL), LiOH (11 g, 260 mmol) In H₂O (200 mL) was added, followed by MeOH (200 mL). The mixture was kept stirring at room temperature for 20 hours. Upon completion of the reaction, 4 N HCl in H₂O was added to adjust pH to 7 at0° C. The mixture was extracted with EtOAc (2×400 ml). The combined organic layer was washed with brine, dried (Na₂SO₄) and concentrated in vacuo to give compound 3 as a yellow solid (11 g, 93%). LC/MS=911.52(M⁺+1). ¹H NMR (300 MHz, CD₃OD) δ 7.95 (d, 1H), 7.90 (s, 1H), 7.48 (s, 1H), 7.31 (d, 1H), 5.42 (s, 1H), 4.37 (dd, 1H), 4.20 (m, 2H), 3.83-3.58 (m, 7H), 3.80 (m, 2H), 3.30 (m, 2H), 2.45 (m, 1H), 2.27(m, 1H), 1.82 (m, 2H), 1.50 (ra, 1H), 1.33 (m, 2H), 1.18 (m, 1H), 1.05 (m, 8H), 0.90 (m, 3H), 0.76 (m, 11H), 0.14-0.04 (m, 2H)

The intermediate compound 315 was prepared as follows.

a. Preparation of compound 301.

To a dry, argon purged three-neck round bottom flask (1000 mL) were added anhydrous dichloromethane (100 mL) and Et₂Zn (28 ml, 273 mmol) at 0° C. (CAUTION: Source of argon can not be from needle. Use appropriate glass adapter only. A second bubbler can also be attached to the flask to prevent excessive pressure build up.) Cyclopenten-3-ol (10.0 mL, 119 mmol) was then added dropwise (large quantity of ethane gas was produced) to the flask and the reaction mixture was slowed to stir until the evolution of gee had ceased. Diiodomethane (22 mL, 242 mmol) was then added dropwise over a period of 30 minutes. The reaction was allowed to warm to mom temperature and continued to stir overnight under a positive flow of ergon, at which point TLC analysis had indicated complete disappearance of the starting alcohol. The reaction was then diluted with CH₂Cl₂and quenched with 2M HCl (white precipitate should be completely dissolved). The biphasic mixture was poured into a separatory funnel and the organic layer was collected. The solvent was removed under reduced pressure until 100 ml of material containing compound 301 remained.

b. Preparation of Compound 302.

Anhydrous dichloromethane (525 mL) was added to the flask followed by the dropwise addition of triethylamine (34 mL, 245 mmol). The reaction continued to stir at room temperature under a positive flow of nitrogen at which point, disuccinimidylcarbonate (40.7 g, 159 mmol) was added to the flask portion wise. The reaction was allowed to stir until TLC analysis Indicated complete disappearance of the starting material (2-3 days). Upon completion, the reaction mixture was quenched with 1M HCl (200 mL×2) and washed with H₂O (200 mL×2). The desired material was extracted using CH₂Cl₂ and the combined organic layers were dried using anhydrous MgSO₄ and passed through a silica plug. The solvent was removed under reduced pressure and the crude material was purified using flash chromatography (R_(t)=0.33, 1:1 Hex/EtOAc) to provide compound 302 (22 g, 75%): ¹H NMR (300 MHz, CDCl₃): δ 5.24 (t, 1H), 3.82 (s, 4H), 2.24 (m, 2H), 2.03 (d, 2H), 1.38 (m, 2H), 0.48 (m, 1H), 0.40 (m, 1H).

c. Preparation of Compound 304.

N4-t-Boc-cis-4-Hydroxy-L-Proline methyl ester 303 (100.0 g, 407.7 mmol) and DABCO (1.5 eq, 68.6 g, 611.6 mmol) were dissolved in anhydrous toluene (200 mL) in a 2 L three necked round bottom flask with a mechanical stirrer and an addition funnel. After cooling the solution to 0° C. under N₂. A solution of 4-Bromo-benzylzenesulfonyl chloride (1.3 eq, 135.6 g, 530.0 mmol) in 300 ml of toluene was added through addition funnel over 60 minutes. The reaction mixture was stirred and warmed to room temperature overnight (16 hours). The mixture was slowly poured into 2L 1M Na₂CO₃ (aq.), and the product was extracted with EtOAc (2L). After the organic phase was washed by 0.5 N HCl (2L), H₂O (1L), and brine (1L), it was dried (MgSO₄), concentrated to give 195.45 g of a yellow oily brosylate product.

To a solution of the above brosylate (407.7 mmol) in dichloromethane (300 mL) was slowly added 4.0 M HCl in dioxane (500 ml, 5 eq) and the resulting solution was allowed to stir at room temperature for 2 hours. After ether (500 mL) was added to the reaction mixture, the mixture was stirred for 18 minutes and the white precipitate was collected by filtration. The solid was washed with ether and hexane and then dried under vacuum overnight to obtain 153.0 g of the HCl amine salt of compound 304, 381.8 mmol, in 84% yield for two steps.

d. Preparation of Compound 305.

To a solution of Boc-tert-butyl-glycine (97.0 g, 420.0 mmol) in DMF (200 mL) and methylene chloride (200 mL) were added HATU (217.76 g, 572.7 mmol) and Hunig's base (126 ml, 1145.4 mmol) at room temperature. After the mixture was stirred for 20 minutes at room temperature, a solution of the previous HCl salt (153.0 g, 381.8 mmol) and Hunig's base (126 mL, 1145.4 mmol) in BMP (200 mL) and dichloromethane (200 mL) was added to the above acid mixture in one portion. The reaction mixture was stirred at room temperature for 3 h, with monitoring by LCMS. The reaction mixture was concentrated to remove dichloromethane under reduced pressure and the white solid that formed was filtered off. The remaining DMF solution was diluted with ethyl acetate (1 L), washed successively with 3% LiCl (aq) (3×650 mL), sat'd NH₄Cl (2×500 mL), 0.5N HCl (aq) (2×600 mL), brine (500 mL), sat'd NaHCO₃ (3×500 mL), and brine (800 mL). The resulting organic fraction was dried (MgSO₄) and concentrated to compound 305 (111 g).

a. Preparation of Compound 306.

To a solution or the methyl ester 305 (120 g, 207.6 mmol) in THF (300 mL), MeOH mL) was added a solution of LiOH (26.18 g, 623.4 mmol) in H₂O (150 mL). The solution allowed to stir at room temperature for 4 hours. The mixture was cooled in an ice-bath whi

acidifying with 3N HCl to pH about 5.5, stirred for 10 minutes, and the resulting white solids were collected by filtration. The solids were washed with more water, ether and hexane. The solids were dried under vacuum at 40° C. overnight to give 95.78 g (82%) of the acid 306.

f. Preparation of Compound 307.

To a solution of the carboxylic acid 306 (81.4 g, 144.27 mmol) in DMF (200 mL) and dichloromethane (200 mL) was added HATU (82.3 g, 216.4 mmol) and Hunig's base (47.5 mmol, 432.8 mmol) at room temperature. After the mixture was stirred for 20 minutes at room temperature, a solution of amine (158.7 mmol) and Hunig's base (47.5 mL, 1145.4 mmol) DMF (200 mL) and dichloromethane (200 mL) was added to the above acid mixture in one portion. The reaction mixture was stirred at room temperature for 3 hours and monitored

LCMS. After the mixture was concentrated under reduced pressure to remove dichloromethane, the white solids that formed were filtered off. The remaining DMF solution was diluted with ethyl acetate (600 mL) and successively washed with 3% LiCl (aq) (2×550 sat'd NH₄Cl (500 mL), 1N HCl (aq) (500 mL), sat'd NaHCO₃ (500 mL), and brine (300 mL). The resulting organic fraction was dried (Na₂SO₄) and concentrated to afford compound 307 (1

g. Preparation of Compound 308.

Compound 307 was dissolved in 4N HCl in dioxane (300 mL) at room temperature stirred for 2 hours. It was then concentrated under vacuum, and co-evaporated with dichloromethane (2×200 mL) to dryness. The residue was dissolved in EtOAc (600 mL) as sat'd aq. NaHCO₃ (1L). If was stirred vigorously. After 10 minutes, carbonic acid bicyclo[3.1.0]hex-3-yl ester 2,5-dioxo-pyrrolidin-1-yl ester 302 (41.4 g, 173.1 mmol) was ac

in one portion. After the resulting mixture was stirred for another 30 minutes, the organic la

was collected and washed with brine (500 mL), dried (Na₂SO₄), and concentrated. The crude product was purified by flesh chromatography on silica gel with ethyl acetate/hexane to aff

94.44 g (92%) of compound 308.

h. Preparation of Compound 310.

1-(2-Amino-3-chloro-4-hydroxy-phenyl)-ethanone 309 (70.7 g, 354 mmol) was stirred in 48% aq. HBr (500 mL) at 110° C. for 72 hours, After the mixture was cooled to 0° C. with stirring, the solids were filtered and washed with water. The resulting solids were triturated with a saturated NaHCO₃ solution (˜350 mL), filtered, washed with wafer, and dried under vacuum to give ˜40 g (61%) of crude 310 as a dark brown solid. LC/MS=186 (M⁺+1).

i. Preparation of Compound 311.

1-(2-Amino-3-chloro-4-hydroxy-phenyl)-ethanone 310 (40 g, 218 mmol) was dissolved In DMF (360 ml). Cesium carbonate (140 g, 430 mmol) was added, followed by bromoacetaldehyde dimethyl acetal (54.5 g, 323 mmol). The mixture was then vigorously stirred at 65° C. for 24 hours. Upon cooling to room temperature, EtOAc (1 L) and H₂O (1 L) were added to the mixture. The organic layer wee extracted with EtOAc (1×400 ml). The combined organic layer was washed with aqueous 3% LiCl solution (2×11), brine, dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel chromatography to give compound 311 as a white solid (39 g, 67%).

j. Preparation of Compound 312.

To a mixture of 1-[2-Amino-3-chloro-4-(2,2-dimethoxy-ethoxy)-phenyl]-ethanone 311 ( 13 g, 47.5 mmol) and isopropylaminothiazole-4-carboxylic acid hydrobromide (12.64 g, 47.5 mmol) in pyridine (150 ml) was slowly added phosphorus oxychloride (9.47 g, 61.8 mmol) at −40° C. The mixture was then stirred at 0° C. for 4 hours, Upon completion of the reaction, H₂O (30 ml) was added dropwise to the mixture. The mixture was then stirred at 0° C. for another 15 minutes. The mixture was concentrated in vacuo. The residue was diluted with EtOAc, washed with a sat, NaHCO₃ aqueous solution. The organic layer was dried (Na₂SO₄ and concentrated in vacuo. The residue was dissolved in CH₂Cl₂, hexanes were added slowly to the solution, and a yellow solid started to crash out. More hexanes were added until not much product was left in the mother liquid to provide compound 312 (18 g, 85%).

k. Preparation of Compound 313.

2-Isopropylamino-thiazole-4-carboxylic acid [6-acetyl-2-chloro-3-(2,2-dimethoxy-ethoxy)-phenyl]-amide 312 (18 g, 40.7 mmol) was suspended in toluene (400 mL). NaH (2.4 g, 61 mmol) was added to the vigorously stirred mixture while monitoring H₂ evolution. The mixture became a clear solution during heating to reflux. The reaction was complete after refluxing for 3 hours. The mixture was cooled to room temperature. A solution of AcOH (69.2 mmol) in H₂O (3 vol) was added to the mixture. After vigorous agitation for 1 hour at 0° C., the solids were collected by filtration, rinsed forward with H₂O. The wet cake was dried under high vacuum to a constant weight to provide compound 313 (15 g, 86%).

l. Preparation of Compound 314.

To a mixture of brosylate intermediate 303 (15 g, 35 mmol) and compound 313 (27.5 g, 38.5 mmol) in NMP (200 mi) was added cesium carbonate (25.1 g, 77 mmol). The mixture was stirred at 65° C. for 5 hours. The reaction was cooled to room temperature and EtOAc (600 ml) and an aqueous solution of 3% LiCl (600 mL) were added to the mixture. The organic layer was washed with aqueous 3% LiCl (1×600 mL), brine, dried (Na₂SO₄) and concentrated in vacuo. The residue was purled by silica gel chromatography to give the desired methyl ester as a yellow solid (23.6 g, 75%). LC/MS=900.13 (M⁺+1).

m. Preparation of Compound 315.

Methyl ester 314 (23.8 g, 26 mmol) was dissolved in glacial acetic acid (200 ml), 1.4 N HCl in H₂O (75 ml) wee added to the solution. The mixture was stirred at 60° C. for 1 hour. Upon completion of the reaction, the mixture was concentrated to remove the solvents, coevaporated with toluene (×2) to remove residual acetic acid. The residue was then dissolved in EtOAc (500 mL) and sat. NaHCO₂ aqueous solution (enough to neutralize the mixture) white monitoring CO₂ evolution. The organic layer was washed with brine, dried (Na₂SO₄) and concentrated in vacuo. The residue was further dried under high vacuum for 1 h and used as is for the next step. The crude was dissolved in CH₂Cl₂ (360 mL), morpholine (3.4 g, 39 mmol) and sodium triacetoxyborohydride (7.2 g, 34 mmol) were added to the mixture at 0° C. Then glacial acetic add (0.47 g, 7.8 mmol) was added dropwise to the mixture. The reaction was complete in 10 minutes at 0° C. Sat. NaHCO₃ aqueous solution was added to quench the reaction. After stirring for another 20 minutes, the organic layer was washed with brine, dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel chromatography to give the desired amine product 315 as a yellow solid (12 g, 50%). LC/MS=924.63 (M⁺+1).

Compound 4 can be prepared as described in the following Example.

Example 4 Preparation of Compound 4.

Diastereomeric mixture 414 was dissolved in heptane and isopropanol (70%:30%, 230 mg in 4.5 mL of the mixed solvents) and subjected to chiral column separation under the following conditions:

Column: Chiralcel OD-H, 2×25 cm

Solvent system: 70% heptane and 30% isopropanol

Flow rate: 6 mL/min.

Loading volume per run: 2.5 mL.

Compound 4 had a retention time of 20 minutes. ¹H NMR (300 MHz, CDCl₃): δ 8.0 1H), 7.1-7.3 (m, 5H), 8.83 (d, 1H), 6.71 (d, 1H), 6.09 (brs, 2H), 5.95 (s, 1H), 5.04 (m, 2H), (q, 1H), 4.35-4.52 (m, 2H), 4.00 (m, 2H), 2.74 (m, 1H), 1.40 (d, 3H), 1.2-1.3 (12H), 0.98 (s, ³¹P NMR (121.4 MHz, CDCl₃): δ 2.72 (s). Compound 4 was subsequently recrystalized from MTBE for x-ray quality crystals.

Compound 4a had a retention time 50 min, ¹H NMR (300 MHz, CDCl₃): δ 7.98 (s, 7.1-7.3 (m, 5H). 6.83 (d, 1H), 6.73 (d, 1H), 8.02 (bra, 2H), 5.95 (a, 1H), 8.08 (d, 1H), 5.00

1H), 4,88 (q, 1H), 4.38-4,58 (m, 2H), 3.98 (m, 2H), 2.74 (m, 1H), 1.40 (d, 3H), 1.2-1.3 (12H, 0.99 (s, 3H). ³¹P NMR (121.4 MHz, CDCl₃): δ 2.61 (s).

The intermediate diastereomeric mixture 414 was prepared as follows,

a. Preparation of Compound 402.

To a solution of compound 401 (22.0 g, 54.9 mmol, prepared according to the procedures described in J.O.C, 2004, 6257) in methanol (300 mL) was dropwise added a

chloride (22 mL) at 0° C. using a dropping funnel over a period of 30 minutes and then stirred room temperature for 18 hours. The mixture was concentrated, re-dissolved in ethyl acetate (400 mL), washed with ice-cold 2 N NaOH, and concentrated to dryness, affording the crude methyl ether 402 as an oil. MS=437.2 (M+Na⁺).

b. Preparation of Compound 403.

To a solution of compound 402 in methanol (300 mL) was added 0.5 M sodium methoxide solution in methanol (20 ml, 10 mmol), and stirred for 18 hours at room temperature. The reaction was quenched with 4.0 N HCl solute in dioxane (2.5 mL, 10 mmol). The mixture was then concentrated, affording the crude compound 483. MS=20

(M+Na⁺).

c. Preparation of Compound 404.

A mixture of compound 403, Tritron X-405 (70% in water, 6.0 g), 50% KOH (in wat

g) in toluene (500 mL) was heated to reflux with a Dean-Stark trap attached. After 1 hour collecting 25 mL of water, benzyl chloride (33 g, 260 mmol) was added and continued to re

with stirring for 16 hours. The mixture was then cooled and partitioned between ethyl ace

(400 mL) and wafer (300 mL). The organic layer was washed with water (300 mL), and concentrated. The residue was purified by silica gel column chromatography (20% EtOAc hexanes), affording the methyl ether 404 as an oil (22.0 g, 89% in three steps). ¹H NMR(

MHz, CDCl₃): δ 7.3 (m, 15H), 4.5-4.9 (m, 7H), 4.37 (m, 1H), 3.87 (d, 1H), 3.56 (m, 2H), 3.

(s, 3H), 1.40 (s, 3H).

d. Preparation of Compound 405.

To a solution of 404 (22.0 g, 49.0 mmol) in acetic acid (110 mL) was added 3 M sui

add (prepared by mixing 4.6 g of concentrated sulfuric acid with 24 mL of water) and stirred 70° C. for 8 hours. The mixture was concentrated to a volume of 20 mL, and partitioned between ethyl acetate and ice-cold 2N NaOH. The ethyl acetate layer was concentrated,

purified by silica gel column chromatography (˜35% EtOAc/hexanes), affording compound 405 as an oil (17.0 g, 80%). MS=457.2 (M+Na⁺).

e. Preparation of Compound 406.

To a solution of compound 405 (45 g, 104 mmol) in DMSO (135 mL) was dropwise added acetic anhydride (90 mL, 815 mmol) at room temperature under argon. The mixture stirred for 18 hours at room temperature, and then poured into ice-water (1 L) while stirring. After ice was completely melted (30 minutes), ethyl acetate (500 mL) was added. The orgs layer was separated. This extraction process was repeated three times (3×500 mL). The organic extracts were combined and concentrated. The residue was purified by silica gel column chromatography (20% EtOAc/hexanes), affording compound 406 as an oil (39 g, 88%). ¹H NMR (300 MHz, DMSO-d₆): δ 7.3 (m, 15H), 4.4-4.8 (m, 7H), 4.08 (d, J=7.5 Hz, 1H), 3.75 (dd, J=2,4, 11.4 Hz, 1H), 3.64 (dd, J=5.4, 11.4 Hz, 1H), 1.51 (s, 3H).

f. Preparation of Compound 407.

To a dry, argon purged round bottom flask (100 mL) were added 7-bromo-pyrrolol[2f][1,2,4]triazin-4-ylamine (234 mg, 1.10 mmol) (prepared according to WO2007056170) and anhydrous THF (1.5 mL). TMSCI (276 μL, 2.2 mmol) was than added and the reaction mix stirred for 2 hours. The flask was placed into a dry ice/acetone bath (−78° C.) and BuLi (2.5

4.0 mmol, 1.6M in hexanes) was added dropwise. After 1 hour, a solution of compound 40 (432.5 mg, 1.0 mmol) in THF was cooled to 0 C. and then added to the reaction flask drops After 1 hour of stirring at −78°C., the flask was warmed to 0° C. and sat. NH₄Cl (5 mL) was added to quench the reaction. The organics were extracted using EtOAc (3×10 mL) and t

combined organic layers were dried using MgSO₄. The solvent was removed under reduce pressure and the crude material was purified using flash chromatography (hexanes/EtOAc 500 mg (80 %) of compound 407 was isolated as a mixture of two anomers, LC/MS=567.

+H⁺). ¹H NMR (300 MHz, CDCl₃): δ 7.85 (m, 1H), 7.27 (m, 15H), 7.01 (m, 1H), 6.51 (m, 1H) 4.66 (m, 8H), 4.40 (m, 2H), 3.79 (m, 3H), 1.62 (s, 2′-CH₃ from the one anomer), 1.18 (s, 2-

from the other anomer).

g. Preparation of Compound 408.

To a solution of Compound 407 (1 g, 1.77 mmol) in CH₂Cl₂. (20 mL) at 0° C. was ad

TMSCN (1.4 mL, 10.8 mmol) and BF₃-Et₂O (1 mL, 8.1 mmol). The reaction mixture was st

at 0° C. for 0.5 hours, than at room temperature for additional 0.5 hour. The reaction was quenched with NaHCO₃ at 0° C., and diluted with CH₃CO₂Et. The organic phase was separated, washed with brine, dried over Na₂SO₄, filtered and concentrated. The residue

purified by chromatography on silica gel, eluted with CH₃CO₂Et-hexanes (1:1 to 2:1), to give compound 408 (620 mg, 81%) as an isomeric mixture. MS=576.1 (M+H⁺).

h. Preparation of Compound 409.

To a solution of compound 480 (150 mg, 0.26 mmol) in CH₂Cl₂ (4 mL) at −78° C. was added BCl₃ (2 mL, 1M in CH₂C₂). The reaction mixture was stirred at −78° C. for 1 hour. The reaction was quenched at −78° C. by dropwise addition of TEA (2 mL) and MeOH (5 mL). The mixture was allowed to warm up to room temperature, evaporated, and co-evaporated with MeOH several times. The residue was treated with NaHCO₃ (1 g in 10 mL, H2O), concentration and purified by HPLC to give the desired product compound 409 (48 mg, 60%). ¹H NMR (3

MHz, D₂O): δ 7.74 (s 1H), 6.76 (d, J=5 Hz, 1H), 6.73 (d, J=5 Hz, 1H), 4.1 (m, 1H), 3.9 (m 1H), 3.8 (m, 2H), 0.84 (s, 3H). MS=305.9 (M+H⁺). The other alpha-anomer was also obtained (9 mg, 11%): ¹H NMR (300 MHz, D₂O): δ 7.70 (s 1H), 6.8 (d, J=5 Hz, 1H), 6.7 (d 5 Hz, 1H), 4.25 (d, J=9 Hz, 1H)5 4.07 (m, 1H), 3.85 (m, 1H), 3.7 (m, 1H), 1.6 (s, 3H). MS 306.1 (M+H⁺).

i. Preparation of Compound 412.

Compound 410 (commercially available, 4.99 g, 23.8 mmol) was dissolved in dichloromethane (100 mL) and alanine isopropyl ester hydrochloride 411 (3.98 g, 23.8 mmol) was added. The resulting clear solution was cooled −78° C. for 30 min. Triethylamine (6.63

47.5 mmol) was added dropwise over 15 minutes. The mixture was then allowed to warm room temperature. After 16 hours, the solvent was removed by argon stream. The residue was re-dissolved in MTBE (25 mL) and the insoluble was removed by filtration under argon. The filtrate was condensed by argon stream and the crude product 412 was used for the n

reaction without further purification. ¹H NMR (300 MHz, CDCl₃): 7.1-7.4 (m, 5H), 5.1 (m, 1H) 4.35 (m, 1H), 4.15 (m, 1H), 1.5 (d, 3H), 1.2 (m, 6H). ³¹P NMR (121.4 MHz, CDCl₃): δ 7.8 a

8.4 (2s).

j. Preparation of Compound 413.

To a solute of compound 409 (1.03 g, 3.37 mmol) in trimethyl phosphate (2.0 mL THF (20 mL) was added N-methyl imidazole (1.5 g, 18.3 mmol) at 0° C. A solution of compound 412 (2.5 g, 8.18 mmol) in THF (3 mL) was dropwise added. The resulting mixture was allowed to warm to room temperature over 1.5 hours. The mixture was partitioned between ethyl acetate and water. The ethyl acetate layer was concentrated and the residue was purified by silica gel chromatography (ethyl acetate to 10% ethanol/ethyl acetate), affording 1.15 g (59%) of compound 413 as 1:1 diastereometric mixture at phosphorous. ¹NMR (300 MHz, CDCl₃): δ 8.02 (s, 1H), 7.1-7.4 (m, 5H), 6.8 (2d, 1H), 6.7 (2d, 1H), 6.08 (b 2H), 5.03 (m, 1H), 4.6 (m, 1H), 4.4 (m, 2H), 3.9-4.1 (m, 3H), 1.31 (d, 3H), 1.2 (m, 6H), 0.83 3H), ³¹P NMR (121.4 MHz, CDCl₃); δ 2.78 (s). MS=575.1 (M+H⁺).

k. Preparation of Compound 414.

To a solution of compound 413 (175 mg, 0.305 mmol) in acetonitrile (2 mL) was ad N,N-dimethylformamide dimethyl acetal (41 μL, 0.34 mmol, 1.1 eq.) and stirred at room temperature for 1 hour. The reaction was complete (by LCMS). The mixture was then concentrated to dryness. To the residue were added DCC (250 mg, 1.21 mmol, 4 eq.), acetonitrile (5 mL) and isobutyric add (55 mg, 58 μL, 2 eq.). The mixture was stirred at ro

temperature for 48 hours. Water (0.2 mL) and trifluoroacetic acid (0.1 mL) were added at

and stirred at room temperature for 84 hours. Sodium bicarbonate (300 mg) was added at

° C. The mixture was stirred at room temperature for 0.5 hour and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (5% metha

dichloromethane), affording 144 mg (73%) of compound 414 as 1:1 diastereomeric mixture phosphorus. ¹H NMR (300 MHz, CDCl₃): δ 8.00 (s, 1H), 7.1-7.4 (m, 5H), 6.83 (d, 1H), 6.7 1H), 5.97 (brs, 2H), 5.94 (d, 1H), 5.07 (2d, 1H), 5.01 (m, 1H), 4.68 (m, 1H), 4.4 (m, 2H), 4.

2H), 2.74 (m, 1H), 1.4 (2d, 3H), 1.2-1.3 (12H). 0.96 and 0.99 (2s, 3H). ³¹P NMR (121.4 M-

CDCl₃): δ 2.56 and 2.65 (2s). MS=645.1 (M+H⁺).

Compound 5 can be prepared as described in the following Example.

Example 5 Preparation of 5: 5-(3,3-dimethylbut-1-yn-1-yl)-3-[(cis-4-hydroxy-4-[[(3S)-tetrahydrofuran=3-yloxy]methyl]cyclohexyl)[[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl]amino]thiophene-2-carboxylic acid 5.

5-(3,3-dimethyl-but-1-ynyl)-3-[((1R)-4-methyl-cyclohex-3-enecarbonyl)-(1-oxa-spiro[2.5]oct-6-yl)-amino]-thiophene-2-carboxylic acid methyl ester 508 (132 mg, 0.28 mmol) and (S)-tetrahydro-furan-3-ol 509 (247 mg, 2.8 mmol) in 1-methyl-pyrrolidin-2-one (3 mL)

treated with potassium tert-butoxide (251 mg, 2.24 mmol), seated at heated to 40° C. for

hours. After cooling the mixtures was treated with 2 M HCl until pH 3, partitioned between

acetate and water and separated. The organic layer was washed with 5% lithium chloride solution, water, brine, and dried over sodium sulfate. After filtration and concentration the residue was purified by HPLC with CH₃CN (0.1% TFA)/H₂O (0.1% TFA) to afford 107 mg (7 yield) of compound 5 as a white powder; MS (m/z): 544.0 [M+H]°; HPLC retention time 4.2 min (2-98% acetonitrile: water with 0.05% trifluoroacetic acid).

The intermediate compound 508 was prepared as follows.

a. Preparation of Compound 502.

(S)-3-hydroxy-4,4-dimethydihydrofuran-2-(3H)-one (2.60 g, 20 mmol) and diisopropylamine (5.2 mL, 30 mmol) in dichloromethane (25 mL) was cooled to −10° C. treated dropwise with acryloyl chloride (2.03 mL, 25 mmol) and stirred for 2 h. 1M HCl (20

was added and the organic layer was washed with sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered and concentrated. Flash chromatography (10-

EtOAc, hexanes) afforded 2.09 g (57% yield) of the desired (S)-4,4-dimethyl-2-oxotetrahydrofuran-3-yl acrylate 501 as a clear oil.

(S)-4,4-dimethy-2-oxotetrahydrofuran-3-yl acrylate 501 (2.05 g, 11.1 mmol) in dichloromethane (17.5 mL) and hexanes (2.5 mL) was cooled to −10° C. and treated with titanium tetrachloride (2.2 mL, 1 M in dichloromethane, 2.2 mmol). The yellow solution was stirred for 15 minutes and treated with isoprene (1.67 mL, 16.7 mmol) dropwise over 5 min. After stirring for 2 hours, an additional portion of isoprene (1.67 ml, 16.7 mmol) was added to the reaction mixture was stirred at −10 to 0° C. for 3.5 hours. The reaction mixture was quenched with ammonium chloride (sat. aq.). Water and ethyl acetate: hexanes (1:1) were added. The organic layer was separated and the aqueous layer was extracted again with

acetate:hexanes (1:1). The combined organic layers ware dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography (10-50% EtOAc:Hex

g column) to afford 1.30 g (46% yield) of (R)-((S)-4,4-dimethyl-2-oxotetrahydrofuran-3-yl) 4-methylcyclohex-3-enecarboxylate 502 as a clear oil.

b. Preparation of Compound 503.

(R)-((S)-4,4-dimethyl-2-oxotetrahydrofuran-3-yl) 4-methylcyclohex-3-enecarboxylate (1.30 g, 5.15 mmol) in THF (10 mL), water (1 mL) and methanol (1 mL) was tested with lithium hydroxide monohydrate (2.16 g, 51.5 mmol) and warmed to 50° C. with stirring. After 1 hour reaction mixture treated with 1M HCl. The mixture was exacted with hexanes:THF (10:1), dried over sodium sulfate, filtered and concentrated to 0.738 g (quantitative yield) of (R)-4-methylcyclohex-3-enecarboxylic acid 503 as a white powder.

c. Preparation of Compound 504.

(R)-4-methylcyclohex-3-enecarboxylic acid 503 (371 mg, 2.65 mmol), azeotropically dried by evaporation from toluene, was treated with potassium phosphate tribasic (1.13 g, 7 mmol), suspended in dichloromethane (7.6 mL) and treated with dimethylformamide (4 drop

The reaction mixture was cooled to 0° C. and treated dropwise with oxalyl chloride (0.75 m

mmol). The reaction mixture was allowed to warm to ambient temperature while stirring for

hours. After filtering the solids, the solution was concentrated, treated with hexanes and concentrated again to afford (R)-4-methylcyclohex-3-enecarbonyl chloride 504 as a light y

oil which was used immediately in the next step.

d. Preparation of Compound 506.

(R)-4-methylcyclohex-3-enecarbonyl chloride 504 (2.65 mmol), 5-(3,3-dimethyl-but-ynyl)-3-(1,4-dioxa-spiro[4.5]dec-8-ylamino)-thiophene-2-carboxylic acid methyl ester 505 (

mg, 0.66 mmol) and potassium phosphate tribasic (562 mg, 2.65 mmol) were suspended

dichloroethane (1.7 mL), sealed with a cap and heated to 90° C. After 18 hours, the reaction mixture was cooled and partitioned between ethyl acetate and water. The organic layer was separated and the aqueous extracted again with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated. Flash chromatography (10-40% EtOAc:Hexanes) afforded 220 mg (67% yield) of the desired 5-(3,3-dimethyl-but-1-ynyl)-3-carboxylic acid methyl aster 506 as a beige foam.

e. Preparation of Compound 507.

5-(3,3-Dimethyl-but-1-ynyl)-3-[(1,4-dioxa-spiro[4.5]dec-8-yl)-((1R)-4-methyl-cyclohex-3-enecarbonyl)-amino]-thiophene-2-carboxylic acid methyl ester 506 (219 mg, 0.438 mmol) w

dissolved in THF (3.5 mL) and treated with 4M HCl (1.75 ml, 7.01 mmol). The reaction mixture was heated to 45° C. and stirred 2 h. Ethyl acetate was added and the organic layer was separated then washed with water, sodium bicarbonate (sat aq), water, and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to 0.190 g (95% yield) of the desired 5-(3,3-dimethyl-but-1-ynyl)-3-[((1R)-4-methyl-cyclohex-3-enecarbonyl)-(4-oxo-cyclohexyl)-amino]-thiophene-2-carboxylic acid methyl ester 507 as a white foam.

f. Preparation of Compound 508.

Trimethylsulfoxonium chloride (79 mg, 0.62 mmol) in DMSO (1.5 mL) was treated with sodium hydride (21 mg, 60% oil dispersion, 0.53 mmol) and stirred of ambient temperature 10 min. 5-(3,3-Dimethyl-but-1-ynyl)-3-[((1R)4-methyl-cyclohex-3-enecarbonyl)-(4-oxo-cyclohexyl)-amino]-thiophene-2-carboxylic acid methyl ester 507 in THF (1 mL+0.5 mL)

added dropwise and the reaction mixture was stirred for 45 min. The orange solution was treated with 5% citric acid until pH 3 and partitioned between water and ethyl acetate. The organic layer was separated and the aqueous was extracted again with ethyl acetate. The combined organics were washed with 5% LiCl, water and brine, and dried over sodium sulfate. After filtration and concentration, the residue was purified by flash chromatography (20-75% EtOAc:hexanes) to afford 0.134 g (70% yield) of 5-(3,3-dimethyl-but-ynyl)-3-[((1R)-4-methyl cyclohex-3-enecarbonyl)-1-oxa-spiro[2.5]oct-6-yl)-amino]-thiophene-2-carboxylic acid me

ester 508 as a white powder.

Compound 6, Can be prepared using synthetic methods and intermediates like those described in U.S. Ser. No. 12/779,023 (US 20100310512 A1). Compound 6, Can also be prepared described in the following Example.

Example 6 Preparation of (1-(3-[6-(9,9-Difluoro-7-[2-[5-(2-methoxycarbonylamino-3-methyl-butyryl)-5-aza-spiro[2.4]hept-6-yl]-3H-imidazol-4-yl]-9H-fluoren-2-yl)-1H-benzoimidazol-2-yl]-2-aza-bicyclo[2.2.1]heptane-2-carbonyl]-2-methyl-proply)-carbamic acid methyl ester 6.

3-[6-(9,9-Difluoro-7-[2-[5-(2-methoxycarbonylamino-3-methyl-butyryl)-5-aza-spiro[2.4]hept-6-yl]-3H-imidazol-4-yl]-9H-fluoren-2-yl)-¹H-benzoimidazol-2-yl]-2-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester 614 (115 mg, 0.138 mmol) wee dissolved in methylene chloride (2 mL) and HCl in dioxane (4M, 2 mL) was added and stirring at room temperature was continued. After 20 minutes, all volatiles were removed in vacuo. The crude materiel was used in the next step without further purification. The crude material was dissolved in DMF (1.5 mL) and DIEA (53.4 mg, 0.414 mmol) was added. A solution of 2-(L) Methoxycarbonylamino-3-methyl-butyric acid 611 (24.2 mg, 0.138 mmol), HATU (52.4 mg, 0.138 mmol) and DIEA (17.8 mg, 0.138 mmol) in DMF (1 mL) was added. The reaction was stirred at room temperature. After 20 minutes, the reaction was diluted with EtOAc and was washed with aqueous bicarbonate solution, aqueous LiCl solution (5%), brine, and was dried over sodium sulfate. Filtration and removal of solvents in vacuo gave the crude material, which was purified by RP-HPLC (eluent: water/MeCN w/0.1% TFA) to yield compound 6 (76 mg). LCMS-ESI⁺: calc'd for C₄₉H₅₄F₂N₆O₆: 888.9 (M⁺); Found: 890.0 (M+H⁺). ¹H-NMR: 300 MHz, (dmso-d₆) δ: 8.20-7.99 (m, 8H), 7.73 (s, 2H), 7.37-7.27 (m, 2H), 5.25 (dd, J=7.2 Hz, 1H), 4.78 (s, 1H) 4.54 (s, 1H), 4.16 (m, 1H), 4.02 (m, 1H), 3.87 (m,1H), 3,74 (m, 1H), 3.55 (s, 3H), 3.53 (s, 3H), 2.75 (m, 1H), 2.25 (m, 2H), 2.09-2.04 (m, 2H), 1.88-1.79 (m, 2H), 1.54 (m, 1H), 0.84-8.77 (m, 15H) 0.63 (m, 4H) ppm. ¹⁹F-NMR: 282 MHz, (dmso-d₆) δ: 109.1 ppm [−74.8 ppm TFA].

The intermediate compound 614 was prepared as follows.

a. Preparation of compound 4-Methylene-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2-methyl ester 602.

4-Methylene-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 601 (10.0 g, 44 mmol) was dissolved in MeOH (75 mL) at room temperature and HCl (4M in dioxane, 75 mL) was added. Stirring at room temperature was continued for 4 hours. All volatiles were removed in vacuo and a beige solid was obtained. The crude material was suspended in methylene chloride (100 mL) and N-Methyl morpholine (13.3 g, 132 mmol) was added. The mixture was cooled to 0° C. and benzyl chloroformate (8.26 g, 48.4 mmol) was added while stirring. After 30 minutes, the reaction was warmed to room temperature and the solution was washed with wafer and aqueous HCl (1M). The solution was dried over sodium sulfate. Filtration and evaporation of solvents gave crude product, which was purified by silica gel chromatography (eluent: EtOAc/hexanes) to yield compound 602 (10.2 g). LCMS-ESI⁺: calc'd for C₁₅H₁₇NO₄: 275.3 (M⁺); Found: 276.4 (M+H⁺).

b. Preparation of a Mixture of Compounds 603 and 604.

An oven-dried 3-neck round bottom flask was equipped with a nitrogen inlet adaptor and a 250 mL addition funnel. The third neck was sealed with a septum. The flask was charged with a stir bar, dichloromethane (120 mL) and diethyl zinc (1.0 M in hexane, 118 mL, 118 mmol) then cooled to 0° C. in an ice bath. The addition funned was charged with dichloromethane (40 mL) and fluoroacetic acid (0.1 mL, 118 mmol). After the diethyl zinc solution had cooled to 0° C. (about 25 minutes), the trifluoreacetic acid solution was added dropwise over 20 min to the stirred reaction mixture. After stirring for another 20 min at 0° C., diiodomethane (9.5 mL, 118 mmol) was added slowly over 4 minutes. After another 20 min, 4-methylene-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2-methyl ester 602 (8.10 g, 29.4 mmol) was added in 30 mL dichloromethane by cannula. The flask containing 4-methylene-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2-methyl ester was then rinsed with another 10 mL dichloromethane and this solution was also transferred to the reaction mixture by cannula. The reaction mixture was allowed to warm to RT arid stirred for 110 h (about 5 days) attar which the reagents wore quenched with saturated aqueous ammonium chloride (˜150 mL). The contents of the flask were slowly poured info a 2 L sep funnel containing saturated aqueous sodium bicarbonate (800 mL). The aqueous phase was extracted three times with 300 mL ethyl acetate. The combined organics ware dried over magnesium sulfate and concentrated to provide a mixture of Compounds 603 and 604.

c. Preparation of a Compound 603.

The crude material from sub-part b was dissolved in 3:1:1 THF/water/acetone (165 mL) then treated with N-methylmorpholilne-N-oxide (3.45 g, 29.4 mmol) and osmium tetroxide (4 wt % in water, 5 mL, 0.818 mmol). After stirring at RT for 7 h, the reagents were quenched with 1 M aqueous sodium thiosulfate (˜100 mL). The contents of the flask were then poured into a 1 L sep funnel containing water (˜300 mL). The aqueous phase was extracted three times with 300 mL dichloromethane. The combined organics were dried over magnesium sulfate and concentrated. The crude residue was purified by silica column chromatography (5% to 45% EtOAc/hexane) to provide 5-aza-spiro[2.4]heptane-5,6-dicarboxylic acid 5-benzyl ester 6-methyl ester 603 as a clear oil (5.54 g, 19.15 mmol, 85%) as a clear oil. ¹H NMR (CDCl₃) δ 7.36-7.29 (m, 5H), 5.21-5.04 (m, 2H), 4.58-4.47 (m, 1H), 3.75 (s, 1.5H), 3.60 (m, 1.5H), 03.51-3.37 (m, 2H), 2.32-2.25 (m, 1H), 1.87-1.80 (m, 1H), 0.64-0.51 (m, 4H).

d. Preparation of 5-Aza-spiro[2,4]heptane-5,8-dicarboxylic acid 5-benzyl ester 508,

5-Axa-spiro[2.4]heptane-5,6-dicarboxylic acid 5-benzyl ester 6-methyl ester 603 (244 mg, 0.840 mmol) was dissolved in THF (2.0 mL)/MeOH (1.5 mL), An aqueous solution of LiOH (35.5 mg, 0.84 mmol) was added and stirring at room temperature was continued. After 3 hours, the reaction was neutralised with aqueous HCl (1M) and the organic solvents were removed in vacuo. The crude mixture was diluted with water and EtOAc and the organic layer was collected. All volatiles were removed in vacuo and the crude acid 606 was used without further purification. LCMS-ESI⁺: calc'd for C₁₅H₁₇NO₄: 275.3 (M⁺); Found: 276.3 (M+H⁺).

e. Preparation of a 2,7-Dibromo-9,9-difluoro-9H-fluorene 608.

2,7-Dibromo-fluorene-9-one 607 (4.0 g, 11.6 mmol) was suspended in deoxofluor (12 mL) at room temperature and EtOH (4 drops) was added. The stirred suspension was heated at T=90° C. for 24 hours (CAUTION: Use of deoxofluor at elevated temperatures, as described above, is cautioned as rapid and violent exotherms may occur). The reaction was cooled to room temperature and poured onto ice containing sodium bicarbonate. A solid formed and was collected via filtration. The crude material was taken into EtOAc and was washed with aqueous HCl (1M) and brine. The solution was dried over sodium sulfate. Filtration and evaporation of solvents gave crude product, which was purified by silica gel chromatography (eluent: EtOAc/hexanes) to yield 608 (3.2 g). ¹⁹F-NMR: 282 MHz, (dmso-d₆) δ: −111.6 ppm. Before using the material in the next step, it was exposed as a solution in EtOAc to charcoal.

f. Preparation of 5-Aza-spiro[2.4]heptane-5,6-dicarboxylic acid 5-benzyl ester 6-[2-(7-bromo-9,9-difluoro-9H-fluoren-2-yl)-2-oxo-ethyl] ester 609.

2,7-Dibromo-9,9-difluoro-9H-fluorene 608 (372 mg, 1.04 mmol), Pd(PPh₃)₄ (30.0 mg, 0.026 mmol), PdCl₂(PPh₃)₂ (16.2 mg, 0.026 mmol), As(PPh₃)₃ (5.0 mg) were dissolved in dioxane (10 mL) under an argon atmosphere. Ethoxyvinyl-tributyl tin (376.4 mg, 1.04 mmol) was added. The mixture was heated for 140 minutes at 85° C. (oil bath). The reaction was cooled to room temperature. N-bromo succinimide (177 mg, 1.0 mmol) was added followed by water (2 mL). The reaction was stirred at room temperature for 3 hours, after which the majority of the dioxane was removed in vacuo. The crude reaction mixture was diluted with EtOAc and was washed with water. All volatiles were removed in vacuo. Toluene was added and all volatiles were removed in vacuo for a second time. The crude material was dissolved in DMF/MeCN (2 ml, 1:1) at room temperature. A solution of N-Cbz-4-cyclopropyl (L) proline 606 (0.64 mmol) and DIEA (268 mg, 2.08 mmol) in MeCN (2 mL) was added and stirring at room temperature was continued. After 14 hours, most of the MeCN was removed in vacuo and the crude reaction mixture was diluted with EtOAc. The mixture was washed with aqueous HCl (1M), aqueous LiCl solution (5%), brine, and was dried over sodium sulfate. Filtration and evaporation of solvents gave the crude reaction product, which was purified via silica gel chromatography (eluent EtOAc/hexanes) to yield compound 609 (178 mg). LCMS-ESI⁺: calc'd for C₃₀H₂₄BrF₂NO₅: 596.4 (M⁺); Found: 595.2/597.2 (M+H⁺).

g. Preparation of 6-[5-(7-Bromo-9,9-difluoro-9H-fluoren-2-yl)-¹H-imidazol-2-yl]-5-aza-spiro[2.4]heptane-5-carboxylic acid benzyl ester 610.

5-Aza-spiro[2.4]heptane-5,6-dicarboxylic acid 5-benzyl ester 6-[2-(7-bromo-9,9-difluoro-9H-fluoren-2-yl)-2-oxo-ethyl] ester 609 (172 mg, 0.293 mmol) was dissolved in m-xylenes (6.0 mL). Ammonium acetate (228 mg, 2.93 mmol) was added and the reaction was stirred at 140° C. for 80 minutes under microwave conditions. The reaction was cooled to room temperature and all volatiles were removed in vacuo. The crude material was purified via silica gel chromatography (eluent: EtOAc/hexanes) to yield compound 610 (80.3 mg). LCMS-ESI⁺: calc'd for C₃₀H₂₄BrF₂N₃O₂: 576.4 (M⁺); Found: 575.2/577.2 (M+H⁺).

h. Preparation of (1-[6-[5-(7-Bromo-9,9-difluoro-9H-fluoren-2-yl)-¹H-imidazol-2-yl]-5-aza-spiro[2.4]heptane-5-carbonyl)-2-methyl-propyl)-carbamic acid methyl ester 612.

6-[5-(7-Bromo-9,9-difluoro-9H-fluoren-2yl)-¹H-imidazol-2-yl]-5-aza-spiro[2.4]heptane-5-carboxylic acid benzyl ester 610 (800 mg, 1.38 mmol) was dissolved in methylene chloride (15 mL) and HBr in AcOH (37%, 2 mL) was added and stirring at room temperature was continued. After 180 minutes, the suspension was diluted with hexanes and the solid was collected via filtration and was washed with hexanes and subjected to vacuum. The erode material was used in the next step without further purification. The crude materiel was dissolved in DMF (4.0 mL) and DIEA (356 mg, 2.76 mmol) was added. A solution of 2-(L)-Methoxycarbonylamine-3-methyl-butyric acid 611 (242 mg, 1.38 mmol), HATU (524 mg, 1.38 mmol) and DIEA (178 mg, 1.38 mmol) in DMF (1 mL) was added. The reaction was stirred at room temperature. After 50 minutes, the reaction was diluted with EtOAc and was washed with aqueous bicarbonate solution, aqueous LiCl solution (5%), brine, and was dried over sodium sulfate. Filtration and removal of solvents in vacuo gave the crude material, which was purified by silica gel chromatography (eluent: EtOAc/hexanes) to yield the slightly impure compound 612 (878 mg). LCMS-ESI⁺; calc'd for C₂₉H₂₉BrF₂N₄O₃: 599.5 (M⁺); Found: 598.5/600.5 (M+H⁺).

i. Preparation of 3-[6-(9,9-Difluoro-7-[2-[5-(2-methoxycarbonylamino-3-methyl-butyryl)-5-aza-spiro[2.4]hept-6-yl]-3H-imidazol-4-yl)-9H-fluoren-2-yl)-¹H-benzoimidazol-2-yl]-2-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester 614.

(1-(6-[5-(7-Bromo-9,9-difluoro-9H-fluoren-2yl)-¹H-imidazol-2-yl]-5-aza-spiro[2.4]heptane-5-carbonyl]-2-methyl-propyl)-carbamic acid methyl ester 612 (840 mg, 1.4 mmol), 3-[6-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-¹H-benzoimidazol-2-yl]-2-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester 613 (615 mg, 1.4 mmol), Pd(PPh₃)₄ (161 mg, 0.14 mmol), K₂CO₃ (579 mg, 4.2 mmol), were dissolved in DME (15 mL)/water (3 mL) under an argon atmosphere. The mixture was heated for 120 minutes at 85-90° C. (oil bath). After 120 minutes additional boronate ester (61 mg, 0.14 mmol) was added and heating was continued. After 3 hours, the reaction was cooled to room temperature. Most of the DME was removed in vacuo and the crude reaction mixture was diluted with EtOAc. The mixture was washed with brine and was dried over sodium sulfate. Filtration and evaporation of solvents gave the crude reaction product, which was purified via silica gel chromatography (eluent: EtOAc/hexanes) to yield compound 614 (878 mg). LCMS-ESI⁺: calc'd for C₄₇H₆₁F₂N₇O₅: 831.9 (M⁺); Found: 832.7 (M+H⁺).

The intermediate compound 613 can be prepared as follows

j. Preparation of 3-(2-Amino-4-bromo-phenylcarbamoyl)-2-aza-bicyclo[2.2.1]heptane-carboxylic add tert-butyl ester 617.

To a solution of 2-Aza-bicyclo[2.2.1]heptane-2,3-dicarboxylic acid 2-tert-butyl ester (0.327 g, 1.36 mmol, 1 eq.), 4-Bromo-benzene-1,2-diamine 615 (0.507 g, 2.71 mmol, 2 eq.) 4-methylmorpholine (0.299 mL, 2 eq.) in 10 ml DMF was added HATU (0.843 g, 1.05 eq.), reaction mixture was stirred at room temperature for 1 hour than concentrated. The reaction mixture was diluted with ethyl acetate and washed with diluted NaHCO₃ aqueous solution

brine. The organic layer was concentrated down and purified by flash column chromatogra

(silica gel, 20 to 80% ethyl acetate/hexane) to give a mixture of regioisomer 3-(2-Amino-4-bromo-phenylcarbamoyl)-2-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester 617.

k. Preparation of 3-(6-Bromo-¹H-benzoimidazol-2-yl)-2-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester 618.

The above mixture of regioisomer 3-(2-Amino-4-bromo-phenylcarbamoyl)-2-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester 617 was dissolved in ethanol and heated to 130° C. in sealed tube overnight and continue heating at 170° C. for 3 days. LC-MS shows desired product and Boc cleaved product (about 1:1 ratio). The mixture was concentrated down and dissolved HCL. Di-tert-butyl dicarbonate (0.6 eq.) was added and reaction was stirred overnight at room temperature. The reaction mixture was concentrated down and purified by flash column chromatography (silica gel, 20 to 80% ethyl acetate/hexane) to give (6-Bromo-¹H-benzoimidazol-2-yl)-2-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl

618 (0.383 g, 72%) as an orange foam.

l. Preparation of Compound 613

A mixture of 3-(6-Bromo-¹H-benzoimidazol-2-yl)-2-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester 618 (264 mg, 0.673 mmol), benzene-1,4-diboronic acid dipl

ester (5 eq., 3.36 g, 6.95 mmol), tetrakis(triphenylphosphine) palladium (5%, 39 mg) and 2

potassium carbonate aqueous solution (3 aq., 1.01 mL) in 5 ml DME was heated to 90° C. under Ar for 4 hours. The reaction mixture was cooled and diluted in ethyl acetate and wa

with saturated sodium bicarbonate solution. The organic layer dried (MgSO4), concentrate and purified by flash column chromatography (silica gel, 20 to 60% ethyl acetate/hexane)

give 3-[6-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-¹H-benzoimidazol-2-yl]-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester 613 (295 mg, yield 85%). LCM ESI⁺: calc'd for C₃₀H₃₆BN₃O₄: 515.45; Found: 516.1 (M+H⁺).

Compound 7 can be prepared using synthetic methods and intermediates like those described in U.S. Pat. No. 7,429,572. Compound 7 can also be prepared as described in the following Example.

Example 7 Preparation of Compound 7.

To an ice-cold suspension of compound 701 (970 g, 3.74 mol) and DMAP (50 g, 0.

mol) in THF (10 L) is added TEA (2.3 kg, 16.5 mol) and water (7 L) which produces a clear solution. Isobutyryl chloride (3 equivalents) is added slowly to the stirmd mixture while maintaining the temperature at about 0° C. An additional 1.2 then 0.7 equivalents of isobut

chloride is added until the HPLC indicates the reaction had proceeded essentially to compl

(a total of about 1.95 kg). The reaction mixture is acidified with concentrated HCl to a pH s about 8.4 and the organic phase is washed with EtOAo (2×10 L). The combined extracts washed with water (1×15 L). The organic phase is filtered and concentrated in vacuo. The residue is dissolved in IPA (ca. 20 kg) and heptane (14.2 kg) is added. The solution is heated to about 74-75° C. to produce a dear solution, then about 5L is removed by distillation. The resulting solution is cooled slowly to RT. A precipitate is formed at about 42-43° C. Cooling continued slowly to 5° C. then stirred overnight. The resulting solid is filtered and the filtrate washed with IPA/heptane (1:8) mixture (13.4 kg), and dried under vacuum at about 60-70° C. afford 1.285 kg (86.65%) of compound 7 which is 99.45% pure by HPLC.

The intermediate compound 706 can be prepared as follows.

a. Preparation of Compound 701.

To a suspension of cytidine (100 g, 0.411 mol) in DMF (2.06 L) is added benzoic anhydride (102.4 g, 0.452 mol). The mixture was stirred at room temperature for 20 hours. DMF was removed in vacuo and the residue was triturated with diethyl ether. The resulting solid was collected by suction filtration and washed with diethyl ether (2×200 mL). Further drying in vacuo at room temperature gave the N⁴ benzamide (140.6 g, 98.3%). A portion of material (139.3 g, 0.401 mol) was dissolved in anhydrous pyridine (1.2 L) and was treated 1,3-dichloro-1,1,3,3-tetraisopropyl-disiloxane (141.4 mL, 0.441 mol) at room temperature, solution was stirred at room temperature overnight. The mixture was concentrated to near dryness in vacuo and coevaporated with toluene (3×200 mL). The residue was treated with EtOAc (1.8 L) and washed with HCl (2×200 mL, 0.05 N), NaHCO₃ (5%, 2×400 mL). The organic layer was washed dried (Na₂SO₄), filtered, and evaporated to dryness. Compound

(256.5 g, >100%) was isolated as a white foam and used without further purification.

b. Preparation of Compound 702.

Compound 701 (235.5 g, 0.40 mol) was dissolved in dry THF (1.22 L). Anhydrous DMSO (180.8 mL, 2.1 mol) was added and the resulting solution was cooled to between −2 and −15×. Trifluoroacetic anhydride (90.6 mL, 0.64 mol) was added dropwise over 45 ml and the solution was stirred between −20° C. and −15° C. for 2 hrs after which anhydrous triethylamine (223.5 mL, 1.6 mol) was added aver 20 minutes. The crude reaction contain ketone 782 was dissolved in EtOAc (500 mL), and the resulting solution was washed with (3×400 mL), dried (Na₂SO₄) and the solvents were removed in vacuo to give a yellow sol

was purified on a silica gel column eluting with a stepwise gradient of Et₂O (0-60%) in hex

followed by a stepwise gradient of EtOAc (50-100%) in hexanes. The crude ketone so-called obtained (˜192 g) was crystallised from petroleum ether to give ketone 702 (138.91 g, 57.5

from cytidine) as a white solid and 22 g of unreacted starting material, 701, as a yellow sol

.

c. Preparation of Compound 703.

Compound 702 (48.57 g, 8.26 mmol) was dissolved in anhydrous toluene (˜400 ml

and the solvent was removed in vacuo with exclusion of moisture. The residue was than freeze dried in vacuo (oil pump) for another 2 hours. With strict exclusion of moisture, the residual foam was dissolved in anhydrous diethyl ether (1.03 L) under argon. The resulting solution is cooled to −78° C. under argon and MeLi (1.6 M, 258.0 mL, 0.413 mol) was added dropwise additional funnel. After the addition was complete, the mixture was stirred for 2 hours at −7

Aqueous 1M NH₄Cl (500 mL) was added slowly. After warming to room temperature, the mixture was washed with H₂O (2×500 mL), dried (Na₂SO₄), and then concentrated to dry

to give a brown foam (˜60 g, >100%).

The reaction was performed two more times using 37.62 g and 56.4 g of compound 702. The combined crude products (126.0 g, 0.212 mol) were dissolved in THF (1.28 L) and treated with concd HOAc (23 mL, 0.402 mol). To the solution was added TBAF (384.0 mL in THF). The solution was stirred at room temperature for 0.75 hours and the mixture was treated with silica gel (750 g) and concentrated to dryness. The powder was placed on a s gel column packed in CH₂Cl₂. Elution with 1:7 EtOH-CH₂Cl₂ afforded a dark waxy solid that pre-adsorbed on silica gel (300 g) and chromatographed as before. Compound 703 (46.4

53.0 % from 702) was isolated as an off-white solid. ¹H NMR (DMSO-d₆): δ 1.20 (s, 3H, C

3.62-3.69 (m, 2H,), 3.73-3.78 (m, 2H,), 5,18 (t, 1H, J=5.4 Hz, OH-5′), 5.25 (s, 1H, OH-2′), (d, 1H, J=5.0 Hz, OH-3′), 5.99 (s, 1H, H-1′), 7.32 (d, 1H, J=5.B Hz), 7.50 (Ψt, 2H, J=7.7 7.62 (Ψ, 1H, J=7.3 Hz), 8.00 (d, 2H, J=7.3 Hz), 8.14 (d, 1H, J=6.9 Hz), 11.22 (s, 1H, NH) Anal. Calcd for C₁₇H₁₉N₃O₁₅·0.5 H2O: C, 55.13; H, 5.44; N, 11.35. Found: C, 55.21; H, 5.4

11.33.

d. Preparation of Compound 704.

Compound 703 (46.0 g, 0.13 mol) was dissolved in anhydrous pyridine and concentrated to dryness in vacuo. The resulting syrup wee dissolved in anhydrous pyridine under argon and cooled to 0° C. with stirring. The brown solution was treated with benzoyl chloride (30 mL, 0.258 mol) dropwise over 10 minutes. The ice bath was removed and stirred continued for 1.5 hours whereby TLC showed no remaining starting material. The mixture quenched by the addition of water (5 mL) and concentrated to dryness. The residue was dissolved in a minimal amount of CH₂Cl₂ and washed with satd NaHCO₃ (1×500 mL) and (1×500 mL). The organic phase was dried (Na₂SO₄) and filtered, concentrated to dryness chromatographed on silica gel eluting with a stepwise gradient of EtOAc-hexanes (25-60%) provide compound 704 as yellow foam (48.5 g, 67%), ¹H NMR (CDCl₃): δ 1.64 (s, 3H, CH

4.50 (m, 1H, H-4), 4.78-4.85 (m, 2H, H-5′, 5a′), 5.50 (d, 1H, J=3.4 Hz, H-3′), 6.42 (s, 1H, H

7.44-7.54 (m, 7H, Ar), 7.57-7.66 (m, 3H, Ar), 7.94 (d, 2H, J×7.8 Hz), 8.05-8.09 (m, 4H, Ar) 8.21 (d, 1H, J=7.3 Hz). Anal. Calcd for C₃₁H₂₇NO₈: C, 65.37; H, 4.78; N, 7.38, Found: C, 65.59; H, 4.79; N, 7.16.

e. Preparation of Compound 705.

Compound 704 (7.50 g, 0.013 mol) was dissolved in anhydrous toluene (150 mL)

argon and cooled to −20° C., DAST (2.5 mL, 18.9 mmol) was added slowly and the cooling was removed after the addition was complete. Stirring was continued for 1 hours and the mixture was poured into satd NaHCO₃ (100 mL) and washed until gas evolution ceased. The organic phase was dried (Na2SO₄), concentrated, and purified by silica gel chromatographer eluting with 1:1 EtOAc-hexanes. Yield was 1.22 g (16.3%) of pure 705 as a white solid, m

241° C. (CH₂Cl₂-hexanes). ¹H NMR (CDCl₃)): δ 1.49 (d, 3H, J=22.4 Hz, CH₃), 4.64 (dd, 1H, 3.44, 12.9 Hz, 4.73 (d, 1H, J=9.5 Hz, H-4′) 4.90 (dd, 1H, J=2.4, 12.7 Hz, H-5a′), 5

(dd, 1H, J=8.8, 20.7 Hz, H-3′), 6.52 (d, 1H, J=18.0 Hz, H-1′), 7.47-7.57 (m, 7H, Ar), 7.62 (m, 3H, Ar), 7.89 (d, 2H, J=6.9 Hz), 8.07-8.11 (m, 5H, Ar), 8.67 (bs, 1H, NH). ¹⁰F NMR (CDCl₃)): δ 3.3 (m). Anal. Calcd for C₃₁H₂₆FN₃O₇.0.7 H₂O: C, 63.74; H, 4.72; N, 7.20. Found: C, 63.71; H, 4.54; N, 7.20.

f. Preparation of Compound 706.

Compound 705 (6.30 g, 0.011 mol) was suspended in methanolic ammonia (ca 7 M

mL) and stirred at room temperature overnight The solvent was removed in vacuo, co-evaporated with methanol (1×20 mL), and ore-adsorbed onto silica gel. The white powder was placed onto a silica gel column (packed in CHCl₃) and the column was eluted with 9% EtOH in CHCl₃), then 17% EtOH and finally 25% EtOH in CH Cl₃). Concentration of the fractions containing the product, filtration through a 0.4 μm disk, and lyophilization from was afforded compound 706, 2.18 g (76%). ¹H NMR (DMSO-d₆;): δ 1.17 (d, 3H, J=22.3 Hz, C

3,83 (dd, 1H, J −2.7.13,7 Hz, H-81), 3.70-3,84 (m, 3H, H-3′, H-4′, H-5a′), 5.24 (app s, 1H, C

3′), 5.60 (d, 1H, J=5.4 Hz, H-5′), 5.74 (d, 1H, J=7.71 Hz, H-5), 8.07 (d, 1H, J=18.9 Hz,

7.31 (s, 1H, NH2), 7.42 (s, 1H, NH2), 7.90 (d, 1H, J=7.3 Hz, H-6). ¹⁹F NMR (DMSO-d₆;): 2.60 (m). Anal. Calcd for C₁₀H₁₄FN₃O₄.1.4 H₂O: C, 44.22; H, 5.95; N, 14.77, Found: C, 4 H, 5.63; N, 14.54. Compound 706 (0.10 g, 0.386 mmol) was converted to the hydrochloride salt by dissolving in water (2 mL) and adjusting the pH to approximately 3.0 with 1 M HCl. water was removed in vacuo and the residue was crystallized from aqueous EtOH to give Compound 706 as the hydrochloride salt (71.0 mg). mp 243° C. (dec); ¹H NMR (DMSO-d₆;) 1.29 (d, 3H. J=22.6 Hz, CH₃), 3.65 (dd, 1H, J=2.3, 12.7 Hz, H-5′), 3.76-3.90 (m, 3H, H-3′, H-5a′), 5.96 (d, 1H, J=17.3 Hz, H-1′), 6.15 (d, 1H, J=7.9 Hz, H-5), 6.33 (d, 1H, J=7.9 Hz 6), 8.69 (s, 1.5H, NH), 9.78 (s, 1.5H, NH). ¹⁹F NMR (DMSO-d₆;): δ 1.69 (m). Anal. Calcd f

C₁₀H₁₄FN₃O₄.HCl: C, 40.62; H, 5.11; N, 14.21. Found: C, 40.80; H, 5.09; N, 14.23.

Compound 8 can be prepared using synthetic methods and intermediates like those described in U.S. Ser. No. 12/632,194. Compound 8 can also be prepared as described in the following Example.

Example 8 Preparation of 4-amino-2-n-butoxy-8-[3′-(pyrrolidin-1″-ylmethyl)-benzyl-5,6, tetrahydropteridin-6-one 8. (R=n-butyl)

To a solution of nitre compound 807 (730 mg, 1.5 mmol) in MeOH (10 mL) was ad

Raney Nickel (−200 μL, slurry in H₂O). The reaction vessel was flushed with H₂ and then s

under an H₂ atmosphere for 1.5 hours. The mixture was filtered through celite with CH₂Cl₂

MeOH (1:1). The filtrate was concentrated under vacuum and left on lyophilizer overnight, free base of compound 8 was obtained as a white solid. To obtain the HCl salt of 8, a sample of the filtrate above was spiked with 1.0 M HCl to pH=1-2 and lyophilized. ¹H NMR (CD₃C

300 MHz): δ 7.65 (s, 1H), 7.50 (m, 3H), 4.96 (s, 2H), 4.44 (t, J=7 Hz, 2H), 4.40 (s, 2H), 4.

2H), 3.48 (m, 2H), 3.19 (m, 2H), 2.02-2.17 (m, 4H), 1.74 (m, 2H), 1.45 (m, 2H), 0.94 (t, J=

Hz, 3H)−[HCl salt]. LCMS-ESI⁺: calc'd for C₂₂H₃₁N₆O₂, 411.5 (M+H⁺); Found: 411.3 (M+H⁺)

The intermediate compound 807 was prepared as follows.

a. Preparation of Compound 802.

To a solution of compound 801 (2.46 g, 10.2 mmol) in THF (34 mL) at −20° C. was added Et₃N (3.14 mL, 22.5 mmol) followed by a solution of NH₃ (2.0 M in MeOH, 5.4 mL, 1 mmol). The mixture was stirred while warming to 0° C. for 1.5 h (LC/MS indicated consumption of starting materials). The reaction mixture containing compound 802 was taken forward with work-up.

b. Preparation of Compound 803.

To a solution of 3-((1-pyrrolidinylmethyl)phenyl)methanamine 806 (1.95 g, 10.2 mm THF (34 mL) at 0° C. was added Et₃N (3.14 mmol, 22.5 mmol) followed by methyl bromoac

(1.04 mL, 22.3 mmol) dropwise. The reaction mixture was stirred until LC/MS indicated consumption of starting materials, approximately 2 hours. The mixture containing compound 803 was taken forward without work up.

c. Preparation of Compound 804.

The reaction mixture containing compound 803 was added to the reaction mixture containing compound 802 at 0° C. The reaction mixture was stirred until LC/MS indicated that consumption of compound 802, approximately 45 minutes. A saturated solution of NH₄Cl (

mL) was added. The layers were separated, and the aqueous layer was extracted with EtO (2×30 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated under vacuum. Purification by silica gel chromatography provided 2.11 g of compound 804. NMR (CD₃OD, 300 MHz); δ (ppm) 7.32-7.16 (m, 4H), 4.69 (s, 2H), 4.19 (q, J=7 Hz, 2H), 4(s, 2H), 3.60 (s, 2H), 2.49 (m,4H),2.40 (s, 3H), 1.78 (m, 4H), 1.23 (t, 3 H, J=7 Hz). LCMS ESI⁺: calc'd for C₂₁H₂₉N₆O₄S: 461.2 (M+H⁺); Found: 461.0 (M+H⁺).

d. Preparation of Ethyl-N-[4-amino-2-methanesulfonyl-5-nitropyrimidin-6-yl], N_(a)-[3′-pyrrolidin-1′-ylmethyl)-benzyl]-glycinate 805.

To a solution a suspension of the sulfide 804 (3.68 g, 8.00 mmol) in EtOH (40 mL) 0° C. was added sodium tungstate dihydrate (792 mg, 2.40 mmol), acetic acid (4.6 mL, 80 mmol), and hydrogen peroxide (3.4 ml, −40 mmol, 35% w/w in H₂O) sequentially. After 3 hours, additional acetic acid (4.6 mL) and hydrogen peroxide (3.4 mL) were added. The reaction was maintained of 0° C. for 18 hours, A saturated solution of Na₂SO₃ (50 mL) was added carefully while at 0° C. followed by CH₂Cl₂ (75 mL). The layers were separated, and aqueous layer was extracted with CH₂Cl₂ (4×50 mL). The combined organic layers were c

over MgSO₄, filtered, and concentrated under vacuum to provide a material containing compound 805 that was used without further purification.

e. Preparation of Compound 807. (R=n-butyl)

To a solution of sulfone 885 (1.0 g, 2.0 mmol) in n-butanol (10 mL) was added TFA (470 μL, 6.1 mmol). The reaction was stirred at 100° C. for 1 hour. The reaction mixture was poured onto a saturated solution of NaHCO₃ (20 mL) and CH₂Cl₂ (30 mL). The layers were separated, and the aqueous layer was extracted with CH₂Cl₂ (30 mL). The combined organ layers were dried over MgSO₄, filtered, and concentrated under vacuum. Purification was conducted by silica gel chromatography (1 g substrate/10 g SiO₂) (2-15% MeOH/CH₂Cl₂) to provide compound 807.

Example 9 Preparation of Compound 9 (from US2010/0296257)

Preparation of (S)-2-{[(1R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-4-(R)-fluoro-3-hydroxy-4-methyl-tetrahydro-furan-2-yl-methoxy]-phenoxyphosphorylamino]-propionic acid isopropyl ester (from US2010/0296257 Example 2)

Synonym: 5′-O-(isopropyl-L-alanate, phenyl phosphoramidyl)-2′-deoxy-2′-fluoro-2′-C-methyl-uridine diastereomeric mixture.

A 5 L 3-necked flask was fitted with a mechanical stirrer, brine ice bath, internal thermometer, and a nitrogen atmosphere. The flask was charged with L-alanine isopropyl ester hydrochloride (82.0 g, 0.490 moles) and anhydrous dichloromethane (0.80 L). While this was stirring, phenyl dichlorophosphate (85.0 g, 0.40 moles) was added in one lot and stirred. While maintaining the internal temperature between −5 to 5° C., a solution of N-methylimidazole (NMI, 250 g, 3.07 moles) in dichloromethane (250 mL) was added over a period of a half hour. The solution was allowed to stir for 1 h in this temperature range. 2′-Deoxy-2′-fluoro-2′-C-methyl-uridine (3, 80.0 g, 0.307 moles) was added at 0° C. In one portion and then the reaction flask was allowed to warm up slowly in the brine bath. At 1 h, the internal temperature was up to −2° C. TLC (8% Methanol in HCL) at 1 h showed that more than 50% of nucleoside was consumed. The bath was removed and the reaction flask reached ambient temperature over 1 h more. TLC after 3 h and at 5 h total showed 95% of the starting nucleoside was consumed. The reaction mixture was quenched by adding methanol (100 mL) and stirring the reaction for 5 minutes. The reaction mixture was washed with 1M HCl (2×500 mL) followed by saturated sodium bicarbonate solution (2×500 mL). The separated organic layer was dried over anhydrous sodium sulfate (50 g) and filtered. The solution was evaporated under reduced pressure and then under high vacuum to dryness to give the crude product as a viscous oil (170 g). NMRs of the crude product (³¹P and ¹H) were taken. The ³¹P-NMR indicated about 1% of the total phosphorus integration was due to the presence of the 3′ isomer 5.

To the crude product was added anhydrous pyridine (1700 mL). The solvent was evaporated under reduced pressure and then under high vacuum in order to reduce the water content of the crude mixture through co-evaporation. The resulting oil was re-dissolved in anhydrous pyridine (500 mL) and then was added excess t-butyldimethylsilyl chloride (9.0 g, 60 mM). The reaction was stirred at ambient temperature. Reaction progress was monitored by UPLC/MS.

After 3 hours, the 3′ impurity 5 could no longer be detected and the reaction was quenched by the addition of methanol (50 mL).

The reaction wee evaporated under reduced pressure to an oil. The residue was dissolved in ethyl acetate (1.5 L) and washed with 1N HCl (2×500 mL), followed by saturated sodium bicarbonate solution (2×500 mL). The organic layer was dried over anhydrous sodium sulfate (50 g), filtered and evaporated under reduced pressure to give the crude product as a pale yellow oil.

The crude oil was diluted with the same volume of dichloromethane and loaded onto a 2.5 Kg silica gel cartridge n a radial compression module at 100 psi of air pressure. Using a gradient pump at 60 psi and a flow rate of 400 ml/min, the cartridge was washed with methylene chloride (4L) followed by a gradient 1-4% methanol in methylene chloride (48 L). Most of the major impurities (di-(isopropylalanyl) phenyl phosphate, 3′, 5′-bis phosphoramidate, 3′-phosphoramidate-5′-TBDMS adduct (7)) eluted with ˜3% gradient. The desired product eluted between 3 and 4% methanol. The product containing fractions were sorted into two lots. The first contained small amounts of upper impurities and the latter was pure product. The first set of fractions contained small amounts of less polar impurities (upper impurities) such as the 3′,5′-bis phosphoramidate and the di-alanylphenyl phosphate and a mostly the Rp diastereomer and required a second column purification. (The relative terminology, upper vs. lower refers to the elution on normal phase silica-gel chromatography, where the “upper isomer” means the first eluting isomer.) The second set of fractions did not have a significant amount of impurities-just the remaining Rp and mostly the Sp diasterereomers, it was later recombined with the twice-columned fractions. The solvent was evaporated under reduced pressure and the resulting white foam was further dried (0.20 mmHg) for 1 h to give 42 g of the impure lot (4:1 upper vs lower isomer based on ³¹P-NMR) and 38 g of the pure lot (1:3 upper vs lower isomer). The impure lot was recolumned in a similar manner to give 3.8 g of 97% pure upper isomer (fraction set aside) and 36 g of pure product in a 4:1 ratio. The two main lots ware dissolved in HCL, combined, evaporated under reduced pressure and dried (50° C., 0.2 mmHg, 24 h) to get 74 g (45.7%) of pure product (Compound 9) with a diastereomeric ratio of 48:51, as a white foam, mp about 75-85° C.

In order to produce an amorphous solid of the diastereomeric mixture, 74 g of the white foam was stirred in with t-butyl methyl ether (750 mL) resulting in a partial solution and a gummy solid residue. While stirring, heptanes (750 mL) was added slowly and the suspension was mechanically stirred for 1 hour until most of the gum was converted to a white solid. The solid was scraped up with a spatula and the resulting slurry was filtered. The solid was washed with heptanes (4×50 mL) and dried under vacuum (50° C., 0.2 mmHg, 24 h) to give a white, amorphous powder (64 g) with a broad melting range of ca 70-80° C. ¹H and ³¹P NMR conformed to structure and HPLC showed a purity of 99.8% with a diastereomeric ratio of 46:54 (also confirmed by ³¹P NMR).

Alternative method to mate a sold mixture of Compound 9. After chromatography, the rest was co-evaporated with dichloromethane twice (5 ml/g) and dried for 24 h at 35-40° C., at 45 mTorr. The foam residue was sieved through a 250 micron sensors and further dried un

the same conditions until the residual dichloromethane fell below 400 ppm as measured b

headspace GC. The resulting fine off-white to white amorphous powder has a glass transl

temperature range of 53.7 to 63.5° C.

Characterization of Compound 9 (Mixture of Isomers)

¹H-NMR (CDCl₃) 010.05 (brs, 1H, NH, Sp), 10.00 (brs, 1H, NH, Rp), 7.49 (d, 1H, C6-H, Sp 7.36 (m, 5H, C6-H, Rp, aromatic), 7.23-7.14 (m, 6H, Rp/Sp, aromatic), 6.18 (br d, 2H, Cl′-

Rp/Sp), 5.63 (d, 1H, C5-H, Sp), 5.58 (d, 1H, C5-H, Rp), 5.01 (m, 2H, CH—(CH₃)₂ Rp/Sp), 4

4.33 (m, 8H, C-5-H₂, ala-NH, C3′-OH, Rp/Sp), 4.12 (m, 2H, ala-CHCH₃, Rp/Sp), 4.01-3.85 4H, C3′-H, C4′-H, Rp/Sp), 1.391.22 (m, 12H, all CH₃, Rp/Sp).

³¹P-NMR (CDCl₃) 03.60 (Rp), 3.20 Sp relative to triphenylphosphate at −17.80 ppm. ES-M

M+1 530.2. Elemental Analysis: Calculated % (including 0.29% water as found by Karl Fis analysis) C, 49.75; H, 5.54; N, 7.90 , F, 3.58, P, 5.84. Found %: C, 49.50; H, 5.44; N, 7.85; 3.62; P, 6.05.

Preparation of 2′-deoxy-2′-fluoro-Z′-C-methyluridine (from US2010/0203257 Example 1)

In a 10 L flask, was added 3′, 5-O-dibenozyl-2′deoxy-2′-fluoro-2′-C-methyl-N4-benzoylcytic (500 g, 0.874 mol) and 70% aqueous acetic acid (7.5 L). The solution was heated to reflux (110° C.) for 20 h. TLC indicated a complete reaction (RfO.6 in 5% methanol in dichloromethane (HCL)). The mixture was cooled to ambient temperature and diluted with water (2 L), After stirring for 2 h, the resulting precipitate was collected by filtration and the was rinsed with wafer (5 L) and dried in the atmosphere at ambient temperature for 12 h to afford 360 g (88%). This dibenzoyluridine intermediate was used directly in the next step by adding if all to freshly prepared methanolic ammonia (5.4 L, ca 25%) at 0° C. This temperature was maintained for 3 h and then allowed to warm to 15° C. for 24 h. TLC indicated a compl

reaction (Rf 0.4 in 10% methanol in HCL). The reaction mixture was filtered through a Celi

bed and concentrated under reduced pressure to give the crude product (216 g). The crude product was stirred with ethyl acetate (325 mL) for 3 h at ambient temperature. The resulting solid was collected by filtration and washed with ethyl acetate (216 mL). The solid was dried under vacuum at ambient temperature for 4 h to afford 180 g (78%) of the desired product 98.7% HPLC purity. ¹H-NMR (DMSO-d6) 011.44 (br s, 1H, NH), 7.95 (d, 1H, C-6H), 5.97

1H, C-1′H), 5.64 (d, 1H, C-5H), 3.84-3.77 (m, 3H, C-5′-Ha, C-3′H, C-4′H), 3.63-3.60 (m, 1H C5′-Hb), 1.23 (d, 3H, C-2-CH₃). ES-MS M-I 259.

Example 10 Preparation of Compound 10 (from US2010/0298257)

Direct precipitation of Compound 10 (from US2010/0298257; Example 4): To a stirred so

of L-alanine isopropyl ester hydrochloride (10.5 g, 61.5 mmol, azeotropically dried, two tim

with 50 ml of toluene each time) in dichloromethane (100 mL) was added phenydichlorophosphate (7.5 mL, 50 mmol) at room temperature. The mixture was cooled to 10° C. and then was added a solution of N-Methylimidazole (30.5 mL, 384.3 mmol) in 30

dichloromethane over a period of 30 min. After completion of the addition, the mixture was stirred between −10 and −15° C. for 1 h. To the above mixture was added 2′-deoxy-2′-fluoro

C-methyluridine (10 g, 38.4 mmol) (see US2010/0298257 Example 1) In one lot and the mi

was stirred below −10° C. for 3 h and then slowly allowed to warm to 20° C. (8 h). The mixture was stirred at this temperature overnight (15 h) and then quenched with 10 mL of methane The solvent was evaporated and the residue was re-dissolved in EtOAc (200 mL). The Et

layer was washed with water (100 mL), 1N HCl (3×75 mL), 2% aqueous NaHCO₃ solution

mL) and brine (50 mL). The organic layer was dried over Na₂SO₄, filtered and concentrated. The residue was dried under high vacuum for 2 h to give white foam (22 g).

The above foam was dissolved in 33 mL of HCl and then was added 65 mL of isopropyl et

give a saturated solution. The solution was filtered though a small pad of Celite and the filtrate was stirred with seeds of Compound 10 for 72 h at ambient temperature (about 22° C.-nota

cooling the suspension to 0° C. led to oiling out the crude product). The white solid was fi

washed with isopropyl ether (20 mL) and dried to give 4.58 g (˜85:15 mixture of Compound R isomer at P respectively as determined by ³¹P NMR) of a white powder. The above solid suspended in 23 mL of HCL and then refluxed for 3 h. The mixture was cooled to room temperature and stirred for 15 h. The white solid was filtered, washed with 4.5 mL of cold

and dried under high vacuum at 45° C. to give pure Compound 10, mp 93.9-104.7° C. HPL

purity 99.74% (3.11 g, 15.2% from the uridine nucleoside).

Compound 10: ¹H-NMR (CDCl₃) δ 8.63 (br s, 1H, NH). 7.47 (d, 1H, C6-H), 7.30 (m, 2H, o-aromatic), 7.26-7.18 (m, 3H, m,p-aromatic), 6.18 (br d, IH, Cl′-H), 5.70 (d, IH, C5-H), 5.02 (

CH—(CH₃)₂), 4.53 (m, 2H, C-5′-H₂) 4.11 (d, IH, C3′-H), 3.07 (m, 3H, C3′-OH, C4′-H, ala-CH—CH₃), 3.77 (br s, IH, ala-NH), 1.39 (d, 3H, C2′-CH₃), 1.37 (d, 3H, ala-CH₃) 1.24 (d, 6H, CH—(CH₃)₂).

Example 11 Preparation of Compound 11 (from US 2010/0081628)

Synthesis of 6-Ethoxy-9-((4aR,6R,7R,7aR)-7-fluoro-2-isopropoxy-7-methyl-2-oxo-tetrahyr

2,5-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)-9H-purin-2-yl-amine (Compound 11) (Compound 19, US 2010/0081628)

(2R,3R,4R,5R)-5-(2-Amino-6-ethoxy-purin-9-yl)-4-fluoro-2-hydroxymethyl-4-methyl-tetrahy

furan-3-ol (150 mg, 0.46 mmol) was disserved in anhydrous pyridine (2 ml) at 0° C. A solution 0.45 M IH-tetrazole in acetenitrile (2.55 mL) was added followed by bis (N,N-diisopropylamine ispropylphosphoramidite (0.16 mL, 0.55 mmol, 1.2 eq). The mixture was allowed to slowly warm to ambient temperature ever 3 h. TLC indicated a complete reaction. The reaction was quenched upon the addition of water (0.1 mL). The reaction solution was concentrated one reduced pressure and then the residue was triturated with ethyl acetate (5 mL). The results white precipitate was removed by filtration and the filtrate was concentrated under reduced pressure.

The resulting intermediate cyclic phosphite residue was dissolved in acetonitrile (2 mL) and then treated with t-butyl hydroperoxide (70% in water, 0.19 mL) for 5 h at ambient temperature TLC indicated a complete reaction. The reaction solution was concentrated under reduced pressure and the residue was purified by column chromatography (Analogix using a gradient 0 to 5% IPA in HCL). The two diastereomers (Compound 11 and R-isomer at P) were separable. Fractions containing each diastereomer were separately combined and concentrated under reduced pressure to white solids to give 20 mg of each diastereomer (combined yield 20%).

Compound 11 ³¹P_NMR (162 MHz, DMSO): δ-6.49; ¹H-NMR (400 MHz, DMSO): δ=8.17 (s, 1H), 6.47 (bs: 2H), 6.27 (d, J=21.2 Hz, 1H), 4.73-4. (m, 4H), 4.45 (q, J=7.0 Hz, 2H), 4.27-4.21 (m, 1H), 1.39-1.34 (m, 9H), 1.20 (d, J=22.8 Hz, 3 MS (ESI): m/z 432.4 [M+H]⁺ R-Isomer at P ⁻P_NMR (162 MHz, DMSO): δ-4.68; ¹H-NMR (400 MHz, DMSO): δ=8.15 (s, 1H), 6.63 (s: 2H), 6.27 (d, J=21.2 Hz, 1H), 4.74-4. (m, 4H), 4.48 (q, J=6.4 Hz, 2H), 4.42-4.37 (m, 1H), 1.36 (t J=7.2 Hz, 3H), 1.32 (d, J=3.6 3H), 1.30 (d, J=3.6 Hz, 3H), 1.22 (d, J=22.8 Hz, 3H). MS (ESI): m/z 432.4 [M+H]⁺

The structures for Compound 11 and the R-Isomer at P are represented below.

Synthesis of (2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl methyltetrahydro-furan-3-ol (Compound 16, US 2010/0081628)

To a 500 mL of dry round-bottomed flask was loaded (2R,3R,4R,5R)-5-(2-amino-6-chloro-purin-9-yl)-2-(benzoyloxymethyl)-b 4-fluoro-4-methyltetrahydrofuran-3-yl benzoate (11 g, 20.

mmol). Anhydrous absolute ethanol (210 mL) was added and followed by anhydrous K₂CO

(28.91 g, 209.2 mmol). The suspension was stirred and heated of 75° C. under nitrogen for h. All the starting material was consumed at that time by TLC test. The mixture was cooled to room temperature and solid was filtered out. The filtrate was neutralized by addition of gla

acetic acid (2.52 g) to pH-7 and concentrated under reduced pressure. The residue was dissolved in methanol and mixed with silica gel (15 g). The dried mixture of crude product

silica gel was transferred to an empty cartridge and separated through column chromatog

(Analogix 220 g, gradient of 0 to 15% MeOH in DCM) to afford product (5% MeOH in DCM a white foam solid (3.73 g, 54.5%). A second white solid was isolated from column (10% M in DCM, 1.44 g) and it is a mixture of two dimers of nucleoside. A more polar, third white s

was collected from column (15% MeOH in DCM, 0.47 g) and it is a mixture of trimers of nucleoside. HPLC purity of product 99.94%,

¹H-NMR (DMSO-d6): δ 8.16 (s, IH, 8-H), 6.55 (s, 2H, NH₂), 6.04 (d, IH, C1-H), 5.86 (d, IH, OH), 5.24 (m, IH, 5′-OH). 4.44 (q, 2H, 6-0CH₂), 4.23-4.08 (m, IH, C3′-H), 3.91-3.82 (m, 2H, H and C5′-H_(a)), 3.71-3.66 (m, IH, C5′-Hb), 1.36 (t, 3H, CH₃ of ethyl), 1.06 (d, 3H, C2′-CH₃).

Synthesis of (2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fl 4-methyltetrahydrofuran-3-yl benzoate (Compound 12, US 2010/0081628)

To a 12 L of three-neck round-bottomed flask was charged 6-chloro-2-aminopurine (225.4 1.329 mol). Anhydrous tert-BuOH (4.5 L) was added and the solution was stirred with a mechanical stirrer at ambient temperature. Potassium tert-butoxide (solid, 151.6 g, 1.35 m was added portion-wise under a flow of nitrogen gas while stirring. The mixture was stirred RT for an additional 30 min. To a 5 L round-bottomed flask was loaded the α-bromide (10.

g, 0.451 mol) and 3 L of anhydrous acetonitrile at ambient temperature. The bromide solution was added to the purine base suspension over 1 min at ambient temperature. The 5 L flask was rinsed with acetonitrile (2×1 L) to transfer bromide completely to the reaction mixture. mixture was heated gradually to 50° C. over 2 h with a heating mantle and controller, and stirred for 20 h. The reaction was almost complete as shown by TLC beta (R₁0.28, 30% Et in hexanes). The reaction was quenched by the addition of sat. NH4Cl (200 mL) to form a suspension. The suspended solid was removed by titration through a 3 cm pad of Cellitee i

2.5 L porcelain Buchner funnel. The solid was washed with toluene (3×100 mL). The comb

filtrate was neutralized by adding 6 N HCl solution until pH 7 (approx 220 mL). The mixture concentrated under reduced pressure. When the volume of mixture was reduced to about

third volume, additional precipitated solid was removed by filtration in a similar manner. The filtrate was further concentrated to a volume of about 800 mL. The residue was loaded onto plug column (1.6 kg flash grade silica gel in a 6 L sintered glass Buchner funnel) and elute

suction) with a gradient of 10% ethyl acetate in hexanes (6 L) to remove non-polar impurities 30% ethyl acetate in hexanes to afford a small amount of lactol (6 L), and then 40%-4S% e acetate in hexanes (4 L) to elute the main amount of product. The product containing fracti

were combined, concentrated under reduced pressure and dried under vacuum (0.2 mmHg

h, ambient temp.) to a white foam solid (150.7 g, β/α=14.1 by NMR. ¹H-NMR. (CDCl₃)beta δ=1.33 (d, 22.4 Hz, 2′-C—CH₃), alpha; 1.55 (d, 22 Hz, 2′-C—CH₃).

The product mixture foam was dissolved in methanol (700 mL) at ambient temperature. Upon standing, a solid slowly formed over 2 h. The suspension was cooled in a freezer to −

for 17 h. The resulting white solid was collected by filtration and washed with cold MeOh (−

3×60 mL) and ethyl other (3×100 mL). The solid was dried under vacuum (0.2 mmHg, 24 h ambient temp.) to afford 110.5 g of β-product with excellent de (β/α 99.8:1 by HPLC). The filtrate was partially concentrated (ca. 400 mL) and then diluted with more MeOH (400 mL) while heating to 80° C. The solution was cooled down to ambient temperature, seeded and cooled to −5° C. The second crop was collected, washed and dried in a similar manner to g

more product as a white solid (12.26 g) with similar diastereomeric purity. The mother liquid was concentrated to dryness under reduced pressure (ca. 25 g). The residue was a mixture β and α-isomers. It was subjected to automated silica gel column chromatography (Analog

240 g cartridge, 40% to 50% ethyl acetate in hexanes) to afford 14,52 g of product foam w

was recrystallized from MeOH, washed and dried in a similar manner to afford an additional 8.46 g of product in high purity.

The three solids were judged to be of similar purity and they were combined to give 131.2 g of white crystalline product, (55% from bromosugar, 49% from lactol). Mp 160.5-16

C. HPLC purity 99.5% including 0.20% alpha.

¹-NMR (pure β-anomer, CDCl₃): δ=8.03 (m, 2H, arom.), 7.93 (m, 2H, arom.), 7.88 (s, 1H,

H), 7.60 (m, 1H, arom.), 7.50 (m, 1H, arom.), 7.44 Cm, 2H, arom.), 7.33 (m, 2H, arom,), 6.4 (dd, 1H, CII′-H), 6.12 (d, 1H, C3′-H), 5.35 (s, 2H, NH2), 5.00 (dd, 1H, C5′-Ha), 4.76 (m, 1H

H), 4.59 (dd, 1H, C5′-Hb), 1.33 (d, 3H, CH₃).

Example 12 Preparation of Compound 12 (from US20110015146)

Synthesis of (2S)-isopropyl2-((((2R,3R,4R,5R)-5(2-amino-6-methoxy-9H-purin-9-yl)-4-fluor

3hydroxy-4-methyltetrahydrofuran-2-yl)methoxy) (phenexy) phosphorylamino)propanoate

To a 250 mL dry round-bottomed flask were loaded phenyl dichlorophosphate (2.66 g, 12.6

mmol) and anhydrous dichloromethane (40 mL). The amino ester salt (2.60 g, 15.53 mmol was added to the solution and the mixture was cooled to −5° C. N-Methyl imidazole (7.7 ml

mmol) was then added quickly via a dry syringe at −5° C. and the solution was stirred at −5° for 1 h. The nucleoside (2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol), 3.04 g, 9.7 mmol) was added from a vial in portion at −5° C. and the solid was slowly dissolved in 20 minutes. The reaction temperature was allowed to rise to ambient temperature over 2 h. After 17 h, the reaction was not comp

More reagents were made (from phosphate (2.66 g), aminoester (2.60 g), and N-Methyl imidazole (3.8 mL, 48 mmol)) and added to the reaction mixture at −5° C. The reaction was stirred at room temperature for 2 more hours. The reaction was almost complete as shown TLC result and diluted with 70 mL of dichloromethane. HCl solution (1 N, 70 mL) was added. The aqueous layer was separated and extracted with dichloromethane. The organic layer

washed with saturated NaHCO₃, water, brine and dried over MgSO₄. After removal of the solvent under reduced pressure, the sticky residue was purified through automated column chromatography using a 240 g cartridge and a gradient of 0-8% 2-PrOH in dichloromethane to afford product as a foam solid (4.16 g, 7.14 mmol, 73% yield). HPLC purity 97.4%. NMR spectra of product showed it is a mixture of two diastereoisomors with a ratio of 1.2:1.

¹H-NMR (DMSO-d₆): δ=7.98 (1H, s, 8-H of one isomer), 7.95 (1H, s, 8-H of another isomer), 7.37-7.32 (2H, m, arom-H), 7.22-7.15 (3H, m, arom-H), 6.6 (2H, s, NH₂), 6.11 (1H, d, Cl′-H of one isomer), 6.09 (1H, d, Cl′-H of another isomer), 6.09-5.98 (1H, m, amide NH), 5.88 (1H, d, 3′-OH of one isomer), 5.81 (1H, d, 3′-H of another isomer), 4.85-4.75 (1H, hepta, methine H of iso-propyl), 4.46-4.27 (2H, m, C4′-H, α-H of amino ester), 4.15-4.07 (1H, m, C3′-H), 3.98 (3H, a, OCH₃), 3.62-3.72 (2H, m, C5′-H_(a)and C5′H_(b)), 1.23-1.06 (9H, m, CH₃'s of amino ester), 1.03 (3H, d, C2′-CH₃).

³¹P-NMR (DMSO-d6): 0=4.91 (one isomer), 4.72 (another isomer).

An alternate purification method is to chemically alter the minor 3′ phosphoramidate by-product in order to simplify the chromatographic separation. The crude phosphoramidate product is dissolved in anhydrous pyridine (5 mL/g), and is treated with 0.5 molar equivalents of t-butyldimethylsilyl chloride at ambient temperature to react selectively with the free 5′ primary hydroxyl of the 3′ isomer impurity. Reaction progress can be monitored by LC/MS. Once the 3′ isomer is converted to a 5′-tBDMS-3′-phosphoramidate derivative, the reaction is quenched with methanol (3 eq), concentrated under reduced pressure, partitioned between ethyl acetate and 5% citric acid and then the organic layer is concentrated. The residue is then subjected to chromatography which can now be done with a higher leading and a faster gradient and achieve a higher purity.

Example 13 Preparation of Compound 13 (from US20110015148)

Example 14 Preparation of Compound 14 (From U.S. Pat. No. 7,964,580, Example 5)

Preparation of 2′-Deoxy-2′-fluoro-2′-C-methyluridine-5′-phenyl methoxy-alanyl phosphate)

Phenyl methoxyalaninyl phosphorochloridate (1 g, 6.5 eq) dissolved in 3 mL of THF was added to a mixture of 2′-Deoxy-2′-fluoro-2′-C-methyluridine (0.15 g, 1 eq) and N-methylimidazole (0.3 g, 8 eq) in 3 mL THF with vigorous stirring at room temperature, than the reaction was stirred overnight. Solvent was removed by reduced pressure. The resulting crude product was dissolved in methanol purified by prep-HPLC on a YMC 25×30×2 mm column using a water/acetonitrile gradient elution mobile phase. The acetonitrile and wafer were removed under reduce pressure to give the desired product (50.1 mg, 15.6%). ¹H NMR (DMSO-d₆) δ 1.20-1.27 (m, 6H), 3.58 (d, J=16.0 Hz, 3H), 3.75-3.92 (m, 2H), 4.015-4.379 (m, 2H), 5.54 (t, J=10.2 Hz, 1H), 5.83-5.91 (m, 1H), 6.00-6.16 (m, 1H), 7.18 (d, J=8.0 Hz, 2H), 7.22 (s, 1H), 7.35 (t, J=4.4 Hz, 2H), 7.55 (s, 1H), 11.52 (s, 1H); MS, m/z 502 (M+1)⁺.

Example 15 Preparation of Compound 15 (Example 55, from U.S. Pat. No. 7,964,580)

¹H NMR (DMSO-d₆) δ>1.20-1.44 (m: 12H), 1.60-1.71 (m, 4H), 3.75-4.02 (m, 2H), 3.94-4.02 (m, 1H), 4.19-4.28 (m, 2H), 4.59-4.61 (m, 1H), 5.57 (f, J=8.4 Hz, 1H), 5.85-6.06 (m, 3H), 7.17-7.23 (m,4H), 7.54 (d, J=8.4 Hz, 1H), 11.50 (s, 1H); MS, m/e 587.92 (M+1)⁺

A general procedure for nucleoside phosphoramidata derivatives is reported at column 46 U.S. Pat. No. 7,964,580. A solution of the appropriate phosphorochloridate (6.5 equivalents) in anhydrous tetrahydrofuran (THF) may be added to a mixture of nucleoside (1 equivalent); N-methylimidazole (6 equivalents) in anhydrous THF with vigorous stirring at room temper with the reaction mixture stirred overnight. Solvent may be removed in vacuo and the cruc

purified by column chromatography and/or preparative thin layer chromatography to give t

desired compound.

Example 18 Preparation of Compound 18 (From U.S. Pat. No. 7,429,572)

Synthesis of (2′R)-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine Starting from Cytidine

Step 1: To a suspension of cytidine (100 g, 0.411 mol) in DMF (2.06 L) is added benzoic anhydride (102.4 g, 0.452 mol). The mixture was stirred at room temperature for 20 h. The DMF was removed in vacuo and the residue was triturated with diethyl ether. The resulting solid was collected by suction filtration and washed with diethyl ether (2×200 mL). Further drying in vacuo at room temperature gave the N⁴ benzamide (140.6 g, 98.3%). A portion

material (139.3 g, 0.401 mol) was dissolved in anhydrous pyridine (1.2 L) and was treated

1,3-dichloro-1,1,3,3 -tetraisopropyl-disiloxane (141.4 mL, 0.441 mol) at room temp. The solution was stirred at room temperature overnight. The mixture was concentrated to near dryness in vacuo and coeavaporated with toluene (3×200 mL). The residue was treated with EtOAc (1.8 L) and washed with HCl (2×209 mL, 0.05 M), NaHCO₃ (5%, 2×400 mL). The organic layer was washed, dried (Na₂SO₄), filtered, and evaporated to dryness. Compound 16-1 (Compound 4-1 from U.S. Pat. No. 7,429,572) (256.5 g, >100%) was isolated as a white foam and used without further purification.

Step 2: Compound 16-1 (236.5 g, 0.40 mol) was dissolved in dry THF (1.22 L). Anhydrous DMSO (180.8 mL, 2.1 mol) was added and the resulting solution was cooled to between −20° C. and −18° C. Trifluoroacetic anhydride (90.6 mL, 0.64 mol) was added dropwise over 45 minutes and the solution was stirred between −20° C. and −15° C. for 2 hrs after which anhydrous triethylamine (223.5 ml, 1.6 mol) was added over 20 min. The crude reaction containing ketone 16-2 was dissolved in EtOAc (500 mL), and the resulting solution was washed with H₂O (3×400 mL), dried (Na₂SO₄) and the solvents were removed in vacuo to give a yellow solid that was purified on a silica gel column eluting with a stepwise gradient of Et₂O (0-60%) in hexanes followed by a stepwise gradient of EtOAc (50-100%) in hexanes. The crude ketone so-obtained (˜192 g) was crystallized from petroleum ether to give ketone 16-2 (Compound 4-2from U.S. Pat. No. 7,429,572) (138.91 g, 57.5% from cytidine) as a white solid and 22 g of unreached starting material, 16-1, as a yellow solid.

Step 3: Compound 16-2 (48.57 g, 8.26 mmol) was dissolved in anhydrous toluene (˜400 mL) and the solvent was removed in vacuo with exclusion of moisture. The residue was then further dried in vacuo (oil pump) for another 2 h. With strict exclusion of moisture, the residual foam was dissolved in anhydrous diethyl ether (1.03 L) under argon. The resulting solution was cooled to −78° C. under argon and MeLi (1.6 M, 258.0 mL, 0.413 mol) was added dropwise via additional funnel. After the addition was complete, the mixture was stirred for 2 h at −78° C. Aqueous 1M NH₄Cl (500 mL) was added slowly. After warming to room temperature, the mixture was washed with H₂O (2×500 mL), dried (Na₂SO₄), and then concentrated to dryness to give a brown foam (˜60 g, >100%).

The reaction was performed two more times using 37.62 g and 56.4 g of compound 16-2. The combined crude products (128.0 g, 0.212 mol) were dissolved in THF (1.28 L) and treated with coned HOAc (23 mL, 0.402 mol). To the solution was added TBAF (384.0 mL, 1 M in THF). The solution was stirred at room temp for 0.75 h and the mixture was treated with silica gel (750 g) and concentrated to dryness. The powder was placed on a silica gel column packed in CH₂Cl₂. Elution with 1:7 EtOH-CH₂Cl₂ afforded a dark waxy solid that was pre-adsorbed on silica gel (300 g) and chromatographed as before. Compound 18-3 (Compound 4-3 from U.S. Pat. No. 7,429,572) (46.4 g, 53.8% from 16-2) was isolated as an off-white solid. ¹H NMR (DMSO-d₆): δ 1.20 (s, 3H, CH₃), 3.82-3.88 (m, 2H), 3.73-3.78 (m, 2H,), 5.19 (t, 1H, J=5.4 Hz, OH-5′), 5.25 (s, 1H, OH-2′), 5.52 (d, 1H, J=5.0 Hz, OH-3′) 5.99 (s, 1H, H-1′), 7.32 (d, 1H, J=5.8 Hz), 7.05 (Ψt, 2H, J=7.7 Hz), 7.62 (Ψt, 1H, J=7.3 Hz), 8.00 (d, 2H, J=7.3 Hz), 8.14 (d, 1H, J=6.9 Hz), 11.22 (s, 1H, NH). Anal. Calcd for C₁₇H₁₉N₃O_(6.)0.5 H₂O: C, 55.13; H, 5.44; N, 11.35. Found: C, 55.21; H, 5.47; N, 11.33.

Step 4: Compound 16-3 (46.0 g, 0.13 mol) was dissolved in anhydrous pyridine and concentrated to dryness in vacuo. The resulting syrup was dissolved in anhydrous pyridine under argon and cooled to 0° C. with stirring. The brown solution was treated with benzoyl chloride (30 mL, 0.250 mol) dropwise over 10 min. The ice bath was removed and stirring continued for 1.5 h whereby TLC showed no remaining starting material. The mixture was quenched by the addition of water (5 mL) and concentrated to dryness. The residue was dissolved in a minimal amount of CH₂Cl₂ and washed with satd NaHCO₃ (1×500 mL) and H₂O (1×500 mL). The organic phase was dried (Na₂SO₄) and filtered, concentrated to dryness and chromatographed on silica gel eluting with a stepwise gradient of EtOAc-hexanes (25-60%) to provide compound 16-4 as yellow foam (Compound 4-4 from U.S. Pat. No. 7,428,572) (48.5 g, 67%). ¹H NMR (CDCl₃): δ 1.64 (s, 3H, CH₃), 4.50 (m, 1H, H-4), 4.78-4.85 (m, 2H, H-5′, 5′), 5.50 (d, IH, J=3.4 Hz, H-3′), 6.42 (s, IH, H-1), 7.44-7.54 (m, 7H, Ar) 7.57-7.66 (m, 3H, Ar), 7.84 (d, 2H, J=7.8 Hz), 8.05-8.09 (m, 4H, Ar), 8.21 (d, 1H, J=7.3 Hz). Anal. Calcd for C₃₁H₂₇N₃O₈. C, 65.37; H, 4.78; N, 7.38.

Found: C, 65.50; H, 4.79; N, 7.16.

Step 5: Compound 16-4 (7.50 g, 0.013 mol) was dissolved in anhydrous toluene (150 mL) under argon and cooled to −20° C. DAST (2.5 mL, 18.9 mmol) was added slowly and the cooling bath was removed after the addition was complete,

Stirring was continued for 1 h and the mixture was poured into satd NaHCO₃ (100 mL) and washed until gas evolution ceased. The organic phase was dried (Na₂SO₄), concentrated, and purified by silica gel chromatography eluting with 1:1 EtOAc-hexanes. Yield was 1.22 g (16.3%) of pure 16-5 (Compound 4-5 from U.S. Pat. No. 7,429,572 as a white solid, mp 241° C. (CH₂Cl₂-hexanes); ¹H NMR (CDCl₃): 5 1.48 (d, 3H, J=22.4 Hz, CH₃), 4.84 (dd, 1H, J=3,44,12. Hz, H-5′), 4.73 (d, 1H, J=0.5 Hz, H-4′), 4.90 (dd, 1H, J=2.4,12.7 Hz, H-5a′), 8.88 (dd, 1H, J=8.6, 20.7Hz, H-3′), 6.52 (d, 1H, J=18. Hz, H-1′), 7.47-7.57 (m, 7H, Ar), 7.82-7.71 (m, 3H, Ar), 7.89 (d, 2H, J=6.9 Hz), 8.07-8.11 (m, 5H, Ar), 8.67 (bs, 1H, NH). ¹⁹F NMR (CDC₃): δ 3.3 (m). Anal. Calcd for C₃₁H₂₅FN₃O₇.7 H₂O : C, 83.74; H, 4.72; N, 7.20, Found: C, 83.71; H, 4.54; N, 7.20.

Step 6: Compound 16-5 (8.30 g, 0.011 mol) was suspended in methanolic ammonia (ca 7 N, 150 mnL) and stirred at room temperature overnight. The solvent was removed in vacuo, co-evaporated with methanol (1×20 mL), and pre-adsorbed onto silica gel. The white powder was placed onto a silica gel column (packed in CHCl₃) and the column was eluted with 9% EtOH in CHCl₃, then 17% EtOH and finally 25% EtOH in CHCl₃. Concentration of the fractions containing the product, filtration through a D.4 1am disk, and lyophilization from wafer afforded compound 18-8 (Compound 4-8 from U.S. Pat. No. 7,428,572), 2.18 g (76%). ¹H NMR (DMSO-d₆); δ 1.17 (d, 3H, J=22.3 Hz, CH₃), 3.63 (dd, 1H, J=2.7, 13.7 Hz, H-5′), 3.70-3.84 (m, 3H, H-3′, H-4′, H-5a′), 5.24 (app s, 1H, OH-3′) 5.60 (d, 1H, J=5.4 Hz, H-5′), 5.74 (d, 1H, J=7.71 Hz, H-5), 6.07 (d, 1H, J=18.9 Hz, H-1′), 7.31 (s, 1H, NH₂), 7.42 (s, 1H, NH₂), 7.90 (d, 1H, J=7.3 Hz, H-6). NMR (DMSO-d₆): δ 2.60 (m). Anal. Calcd for C₁₀H₁₄FN₃O₄4.1.4 H₂O: C, 44.22; H, 5.95; N, 14.77. Found: C, 42.24; H, 5.63; N, 14.54. Compound 16-6 (0.10 g, 0.386 mmol) was converted to the hydrochloride salt by dissolving in water (2 mL) and adjusting the pH to approximately 3.0 with 1 M HCl. The water was removed in vacuo and the residue was crystallized from aqueous EtOH to give 16-6 as the hydrochloride salt (71.0 mg). mp 243

(dec); ¹H NMR (DMSO-d₆): δ 1.29 (d, 3H, J=22.6 Hz, CH₃), 3.65 (dd, 1H, J=2.3, 12.7 Hz,

3.76-3.00 (m, 3H, H-3′, H-4′, H-5a”), 5.96 (d, 1H, J=17.3 He, H-1′), 0.18 (d, 1H, J=7.9 Hz, h 8.33 (d, 1H, J=7.9 Hz, H-6) 8.69 (s, 15H, NH), 9.78 (s, 1.5H, NH). ¹⁹F NMR (DMSO-d₄): δ (m). Anal. Calcd for C₁₀H₁₄FN₃O₄.HCl: C, 40.62; H, 5.11; N, 14.21. Found: C, 40.80; H, 5.0 14.23.

Biological Examples Assay Protocol High Throughput Replicon Assay (HTBS)

Replicon cells harboring H77 (genotype 1a) or Con1 (genotype 1b) HCV RNA end Renilla luciferase reporter were seeded in 384-well black plates at a density of 1.6×103 c

per well in 90 μl of DMEM culture medium, excluding G-418, Compounds were serially diluted in 100% DMSO and added to cells at a 1:228 dilution, achieving a final concentration of 0.44% DMSO in a total volume of 90 μL with a Biotek μFlow Workstation. Cell plates were incubated at 37° C. with 5% CO2 for 3 days, after which culture media were removed and a were assayed for luciferase activity as a marker for replication level. Luciferase expression measured using Dual-Glo luciferase assay reagents (Promega, Madison, Wis.). Briefly, 20 μl Dual-Glo luciferase buffer was added to lyse the cells for 10 min and subsequently 20 μL o

diluted Dual-Glo Stop & Glo substrate (1:100) was added to each well. Luminescence sign was measured an a Perkin Elmer Envision Plate Reader after incubation for 10 minute. Luciferase levels were converted into percentages relative to the untreated controls (define 100%) and data were fit to the logistic dose response equation y=a/(1+(x/b)c) using XLFit

software (IDBS, Emeryville, Calif.). EC₅₀ values were calculated from the resulting equations. Alternatively, antiviral activity may be analyzed by HCV NS3 Protease IC₅₀ Determination. HCV HS3 protease activity was monitored using a fluorescence resonance energy transfer (FRET) depsipeptide substrate (RET S1, Anaspec, San Jose, Calif.) based on the method of Taliani, Taliani M, Bianchi E, Narjes F, Fossatelli M, Urbani A, Steinkuhler C, et al. A continuous assay of hepatitis C virus protease based on resonance energy transfer depsipeptide substrates. Anal. Biochem. 1996; 240 (1):60-7, herein incorporated by reference with regard to performing such assay.

Briefly, 2-10 nM of purified NS3 protease domains were pre-incubated at 37° C. for 10 minutes with 20 μM isogenic NS4A peptide cofactors (Sigma, St. Louis, Mo.), in 40% glyce

buffer with 50 mM HEPES pH 7.5 and 10 mM DTT. Compounds were diluted serially 1:3

DMSO, incubated with the enzyme/cofactor for 10 minutes and reactions were sta

by the addition of 2 μM RET S1 substrate (final concentration). Fluorescence increase wa

measured continuously ever one hour using a Victor3 V fluorescence plate reader (Parkin Elmer, Waitham, Mass.). Initial velocities were calculated for each inhibitor concentration using Workout 1.5 software (DAZDAQ, East Sussex, UK) with the maximal slope algorithm Velocity data, were converted into percentages relative to the untreated control (defined as 100%) and non-linear regression was performed to calculate 50% inhibitory concentration (IC₅₀ values).

NS3 Enzymatic Potency: Purified NS3 protease is completed with NS4A peptide and the incubated with serial dilutions of the compounds (DMSO used as solvent). Reactions are started by addition of dual-labeled peptide substrate and the resulting kinetic increase in fluorescence is measured. Non-linear regression of velocity data is performed to calculate IC₅₀s. Activity is initially tested against genotype 1b protease. Depending an the potency obtained against genotype 1b, additional genotypes (1a, 2a, 3) and or protease inhibitor resistant enzymes (D168Y, D168V, or A156T mutants) may be tested. BILN-2061 is used control during all assays. Compounds of the Examples were evaluated in this assay and

found to have CC₅₀ values of less than about 1 μM.

Replicon Potency and Cytotoxicity: Huh-luc cells (stably replicating Bartenschlager's I389

ubi-neo/NS3-3′/ET genotype 1b replicon) are treated with serial dilutions of compound (DM

is used as solvent) for 72 hours. Replicon copy number is measured by bioluminescence, non-linear regression is performed to calculate EC₅₀s. Parallel plates treated with the same drug dilutions are assayed for cytotoxicity using the Promega CellTiter-Glo cell viability ass

Depending on the potency achieved against the 1b replicon, compounds may be tested ag

a genotype 1a replicon and/or inhibitor resistant replicons encoding D168Y or A156T muta

BILN-2061 is used as a control during all assays. Compounds of the Examples were evaluated in this assay and were found to have EC₅₀ values of less than about 5 μM.

Effect of Serum Proteins on Replicon Potency

Replicon assays are conducted in normal cell culture medium (DMEM+10% FBS) supplemented with physiologic concentrations of human serum albumin (40 mg/mL) or α-a

glycoprotein (1 mg/mL), EC₅₀s in the presence of human serum proteins are compared to EC₅₀ in normal medium to determine the fold shift in potency.

Enzymatic Selectivity: The inhibition of mammalian proteases including Porcine Pancreatic Elastase, Human Leukocyte Elastase, Protease 3, and Cathepsin D are measured at K_(m) fo

respective substrates for each enzyme. IC₅₀ for each enzyme is compared to the IC₅₀ obtained with NS3 1b protease to calculate selectivity.

MT-4 Cell Cytotoxicity: MT4 cells are treated with serial dilutions of compounds for a five period. Cell viability is measured at the and of the treatment period using the Promega CellTiter-Glo assay and non-linear regression is performed to calculate CC₅₀.

Compound Cooperation Associated with Cells at EC₅₀: Huh-luc cultures are incubated

compound at concentrations equal to EC₅₀. At multiple time points (0-72 hours), cells are washed 2× with cold medium and extracted with 85% acetonitrile; a sample of the media

each time-point is also extracted. Cell and media extracts are analyzed by LC/MS/MS to determine the molar concentration of compounds in each fraction.

Solubility and Stability: Solubility is determined by taking an aliquot of 10 mM DMSO stoc

solution and preparing the compound at a final concentration of 100 μM in the test media solutions (PBS, pH 7.4 and 0.1 N HCl, pH 1.5) with a total DMSO concentration of 1%. The test media solutions are incubated at room temperature with shaking for 1 hr. The solution then centrifuged and the recovered supernatants are assayed on the HPLC/UV. Solubility be calculated by comparing the amount of compound detected in the defined test solution compared to the amount detected in DMSO at the same concentration. The stability of compounds after 1 hour incubation in the test media at 37° C. is also determined.

Stability in Cryo-preserved Human, Dog, and Rat Hepatocytes: Each compound is incubated for up to 1 hour in hepatocyte suspensions (100 μl, 80,000 cells per well) at 37° C. Cryopreserved hepatocytes are reconstituted in the serum-free incubation medium. The suspension is transferred into 96-well plates (50 μL/well). The compounds are diluted to 2 incubation medium and then are added to hepatocyte suspensions to start the incubation. Samples are taken at 0, 10, 30 and 60 minutes after the start of incubation and reaction ca

quenched with a mixture consisting of 0.3% formic acid in 90% acetonitrile/10% water. The concentration of the compound in each sample is analyzed using LC/MS/MS. The disappearance half-life of the compound in hepatocyte suspension is determined by fitting concentration-time data with a monophasic exponential equation. The data is also scaled

represent intrinsic hepatic clearance and/or total hepatic clearance.

Stability in Hepatic S9 Fraction from Human, Dog, and Rat: Each compound is incubated

up to 1 hour in S9 suspension (500 μl, 3 mg protein/mL) at 37° C. (n=3). The compounds s added to the S9 suspension to start the incubation. Samples are taken at 0, 10, 30, and 60 minutes after the start of incubation. The concentration of the compound in each sample is analyzed using LC/MS/MS. The disappearance half-life of the compound in S9 suspension determined by fitting the concentration-time data with a monophasic exponential equation,

Caco-2 Permeability: forward (A-to-B) and reverse (B-to-A) permeability is measured Caco-2 monolayers are grown to confluence on collagen-coated, microporous, polycarbons membranes in 12-well Costar Transwell® plates. The compounds are dosed on the apical for forward permeability (A-to-B), and are dosed on the basolateral side for reverse permeability (B-to-A). The cells are incubated at 37° C. with 5% CO₂ in a humidified incubator. At the beginning of incubation, at 1 hr and 2 hr after incubation, a 200-μL aliquot is taken from the receiver chamber and replaced with fresh assay buffer. The concentration of the compound each sample is determined with LC/MS/MS. The apparent permeability, Papp, is calculated Plasma Protein Binding; Plasma Protein Binding is measured by equilibrium dialysis. Each compound is spiked info blank plasma at a final concentration of 2 μM. The spiked plasma phosphate buffer is pieced into opposite sides of the assembled dialysis cells, which is the rotated slowly in a 37° C. water bath. At the and of the incubation, the concentration of the compound in plasma and phosphate buffer is determined. The percent unbound is calculated using the following equation:

${\% \mspace{14mu} {Unbound}} = {100 \cdot \left( \frac{C_{f}}{C_{b} + C_{f}} \right)}$

Where C_(f) and C_(b) are free and bound concentrations determined as the post-dialysis buffer plasma concentrations, respectively.

CYP450 Profiling: Each compound is incubated with each of 5 recombinant human CYP450 enzymes, including CYP1A2, CYP2C9, CYP3A4, CYP2D6 and CYP2C19 in the presence absence of NAOPH. Serial samples can be taken from the incubation mixture at the beginning of the incubation and at 5, 15, 30,45 and 60 min after the start of the incubation. The concentration of the compound in the incubation mixture is determined by LC/MS/MS. The percentage of the compound remaining after incubation at each time point is calculated by comparing with the sampling at the start of incubation.

Stability in Rat, Dog, Monkey and Human Plasma: Compounds are incubated for up for 2 h plasma (rat, dog, monkey, or human) at 37° C. Compounds are added to the plasma at fin

concentrations of 1 and 10 μg/mL. Aliquots are taken at 0, 5, 15, 30, 60, and 120 min after adding the compound. Concentration of compounds and major metabolites at each timep

are measured by LC/MS/MS. Biological data (antiviral potency (EC₅₀) is determined using Renilla luciferase (RLuc)-based HCV replicon reporter assay—HCV 1b RLuc).

Biological Example 1 Anti-HCV Activity of the Combination of Compound 1 and Compound Materials and Methods

Compound 1 and Compound 2 were synthesized by Gilead Sciences (Foster City, Calif.).

Cell Lines

HCV genotype 1b replicon cells (Huh-luc) were obtained from Reblikon (Mainz, Germany). The replicon in these cells is designated I389luc-ubi-neo/NS3-3′/ET and encoded selectable resistance marker (neomycin phosphotransferase) as well as the firefly luciferase reporter gene. Huh-luc cells were maintained in Dulbecco's Modified Eagle's Medium (DM

GIBCO, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, and 0.5 mg/mL of G-418 (GIBCO), Cells were passaged twice a week and maintained at subconfluent levels.

EC₅₀ Determinations

Replicon cells were seeded in 96-well plates at a density of 5×10³ cells per well in 100 μL of DMEM culture medium, excluding G-418. Compounds 1 and 2 were serially dilut

1:3 in 100% DMSO (Sigma). These serial dilutions were added to the cells at a 1:200 dilu

to achieve a final concentration of 0.5% DMSO in a total volume of 200 μL. Plates were incubated at 37° C. for 3 days, after which culture media were removed and cells were lysed assayed for luciferase activity using a commercial luciferase assay (Promega, Madison, Wis.

HCV replication levels in drug-treated samples were expressed as a percentage of those in untreated controls (defined as 100%), and data were fit to the logistic dose response equa

y=a/(1+(x/b)c) using XLFit4 software (IDBS, Emeryville, Calif.). EC₅₀ values were calculated the resulting equations as described previously (Delaney, W. E., et al., Antimicrobial Agents Chemotherapy, 45(6):1705-1713 (2001)).

Antiviral Combination Studies

Replicon cells were seeded in 96-well plates at a density of 5×10³ cells per well in 100 μl of culture medium. Compounds 1 and 2 were serially diluted in 100% DMSO as described above and added in a matrix format to 96-well plates, achieving a defined set of different drug concentrations and ratios in a final volume of 200μL and a final DMSO concentration of 0.5%. For each individual drug, the EC₅₀ value was selected as the midpoint for the concentration range tested. Cells ware incubated for three days and analyzed for luciferase expression as indicated above. For the combination study, two independent experiments were performed in triplicate.

Combination Data Analyse

Data ware analyzed using the MacSynergy II program developed by Prichard and Shipman (Prichard M N, Aseltine K R, Shipman C, Jr., MacSynergy™ II, Version 1.0. University of Michigan, Ann Arbor, Mich., 1983; Prichard M. N., Shipman C, Jr., Antiviral Res 14 (

5):181-205 (1990); Prichard M. N., Shipman C, Jr., Antivir Ther 1 (1):9-20 (1996); Prichard

et al., Antimicrob Agents Chemother 37 (3);540-5 (1993). The software calculates theoretical inhibition assuming an additive interaction between drugs (based on the Bliss Independance model) and quantifies statistically significant differences between the theoretical and observe inhibition values. Plotting these differences in three dimensions results in a surface where elevations in the Z-plane represent antiviral synergy and depressions represent antiviral antagonism between compounds. The calculated volumes of surface deviations are expre in nM²%. Per Prichard and Shipman, combination effects are defined as:

-   -   Highly synergistic if volumes >100 nM².     -   Slightly synergistic if volumes are >50 and ≦100 nM².     -   Additive if volumes are >−50 nM² and ≦50 nM².     -   Slightly antagonistic if volumes are >−100 nM² and ≦−50 nM.     -   Antagonistic if volumes are ≦−100 nM².

Results

Prior to initiating combination experiments, EC₅₀ values in Huh-luc replicon cells were determined for Compound 1 and Compound 2 and results are shown in Table II. Both compounds had an antiviral effect.

TABLE II Individual EC₅₀s for Anti-HCV Compounds 1 and 2 in Huh-luc Replicon Cells Compound EC₅₀ (nM)^(a) Compound 1 3 ± 2 Compound 2 11 ± 3  ^(a)EC₅₀ indicates average ± standard deviation for two or more independent experiments. The antiviral effect of the combination of Compound 1 and Compound 2 was measured the resulting data were analyzed using MacSynergy II, which provides surface plots displa

 significant deviations from additivity. Quantification of statistically significant deviations fro

 additivity indicated that the combination of Compounds 1 and 2 had synergy/antagonism volumes between −50 nM² and 50 nM² indicating additive antiviral effects as shown in Tab

indicates data missing or illegible when filed

TABLE III Quantification of Antiviral Synergy and Antagonism and Drug Interactions for Combinatio Compound 1 and Compound 2 Drug(s) Used in Combination with Synergy Volume Antagonism Compound 2 (nM²)^(a) Volume (nM²)^(a) Interaction Compound 1 13.5 ± 10.5 0.07 ± 0.07 Additive ^(a)Values represent the mean ± standard deviation of two independent experiments perfo

 in triplicate The results of the in vitro experiments set forth in Table III indicate that Compound 2 has additive antiviral activity when combined with Compound 1.

indicates data missing or illegible when filed

Biological Example 2 Combinations with Compound 3 Materials and Methods Antiviral Compounds

Compound 1 and Compound 3 ware synthesized by Gilead Sciences (Foster City, Calif.), Ribavirin and IFn-α were purchased from Sigma (St. Louis, Mo.).

Cell Lines

HCV genotype 1b replicon cells (Huh-luc) were obtained from Reblikon (Mainz, Germany) replicon in these cells is designated I389luc-ubi-neo/NS3-3′/ET and encodes a selectable resistance marker (neomycin phosphotransferase) as well as the firefly luciferase reporter gene. Huh-luc cells were maintained in Dulbecco's Modified Eagle Medium (D-MEM) with GlutaMAX™ (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS, Hycione, Logan, Utah) and 0.5 mg/ml of G-418 (Invitrogen). Cells were passaged twice a

and maintained at subconfluent levels.

EC₅₀ Determinations

Replicon cells were seeded in 96-well plates at a density of 5×10³ cells per well in 100 μl DMEM plus 10% FBS culture medium, excluding G-418. Compounds were serially diluted in 100% DMSO (Sigma). These serial dilutions were added to the cells at a 1:200 dilution achieve a final concentration of 0.5% DMSO in a total volume of 200 μL. Plates were incu

at 37° C. for 3 days, after which culture media were removed and cells were lysed and assayed for luciferase activity using a commercial luciferase assay (Promega, Madison, Wis.), HCV replication levels in drug-treated samples were expressed as a percentage of those in untreated controls (defined as 100%), and data were fit to the logistic dose response equa

y=a/(1+(x/b)^(c)) using XLFit4 software (IDBS, Emeryville, Calif.). EC₅₀ values were calculated the resulting equations as described previously.

Antiviral Combination Studies

Replicon cells were seeded in 96-well plates at a density of 5×10³ cells per well in 100 μL culture medium, excluding G-418. Compound 3 and other compounds were serially diluted 100% DMSO as described above and added in a matrix format to 96-well plates, achieving defined set of different drug concentrations and ratios in a final volume of 200 μL and a final DMSO concentration of 0.5%. For each individual drug (with the exception of Ribavirin), t

EC₅₀ value was selected as the midpoint for the concentration range tested. For Ribavirin, which did not have a selective antiviral affect, a top dose of 6.2 μM was selected since this approximately 3-fold below the concentration at which cytotoxicity started to be observed, were incubated with drugs for three days and analyzed for luciferase expression as indicated above. For each combination study, two independent experiments were performed in tripl

Combination Data Analysis

Data were analyzed using the MacSynergy II program developed by Prichard and Shipman. The software calculates theoretical inhibition assuming an additive interaction between

(based on the Bliss Independence model) and quantifies statistically significant differences between the theoretical and observed inhibition values. Plotting these differences in three dimensions results in a surface where elevations in the Z-plane represent antiviral synergy depressions represent antiviral antagonism between compounds. The calculated volumes surface deviations are expressed in nM²%. Per Prichard and Shipman, combination effect defined as follows:

-   -   Strong synergy if volumes>100 nM²; this amount of synergy is         probably important in vivo     -   Moderate synergy if volumes are >50 and ≦100 nM²; this amount of         synergy may be important in vivo     -   Minor synergy if volumes are >25 and <50 nM²     -   Additivity if volumes are >−25 nM² and ≦25 nM²     -   Minor antagonism if volumes are <−25 and >−50 nM²     -   Moderate antagonism if volumes are >−100 nM² and ≦=50 nM²; this         amount of antago         may be important in vivo     -   Strong antagonism if volumes are ≦−100 nM²; this amount of         antagonism is probably important in vivo

Results EC₅₀ Values for Individual Compounds in Huh-luc Replicon Cells.

Prior to initiating combination experiments, EC₅₀ values in Huh-luc replicon cells were determined for each compound as shown in Table IV. All compounds had an antiviral effe

with the exception of Ribavirin, which had no antiviral activity up to concentrations which was beginning to show cytotoxicity.

TABLE IV Individual EC₅₀s for Anti-HCV Compounds in Huh-luc Replicon Cells Compound EC₅₀ (nM)^(a) Compound 3 2.3 ± 2.6 IFN-α 0.105 ± .003 (U/mL)^(b) Ribavirin >12,500 Compound 1  0.4 ± 0.14 ^(a)EC₅₀ indicates average ± standard deviation for two or more independent experiments. ^(b)INF-α EC₅₀ is expressed in Units (U) per milliliter (mL) instead of a nanomolar concentration.

Combination Antiviral Effects and Drug Interactions

The antiviral effects of Compound 3 when combined with IFN-α, Ribavirin, and Compound

were assayed. The resulting data were analyzed using MacSynergy II, which provides sur

plots displaying significant deviations from additivity. Quantification of statistically significant deviations from additivity indicated that combinations of Compound 3 with resulted in minor synergy (synergy volumes of 32 and 36.5 nM², respectively; Table V). The combination of Compound 3 with the non-nucleoside NS5B inhibitor Compound 1 yielded an synergy volume of 14.5 nM² which indicates an additive antiviral interaction. None of the compound yielded antiviral antagonism volumes outside of the additive range (>−25 nM²) when combine with Compound 3 as shown in Table V.

TABLE V Quantification of Antiviral Synergy and Antagonism and Drug Interactions for Drug Combinations with Compound 3 Drug(s) Used in Combination with Synergy Antagonism Compound 3 Volume (nM²)^(a) Volume (nM²)^(a) Interaction IFN-α   32 ± 4.2 0.15 ± 0.2 Minor synergy Ribavirin   54 ± 14.1  1.6 ± 2.3 Moderate synergy Compound 1 14.5 ± 0.7 4.22 ± 5.0 Additive ^(a)Values represent the mean ± standard deviation of two independent experiments perfo

 in triplicate

indicates data missing or illegible when filed

These in vitro antiviral combination experiments indicate that the novel HCV NS3 protease inhibitor Compound 3 has minor synergy when combined with IFN-60 and moderate synergy when combined with Ribavirin. These results suggest that Compound 3 could potentially be used in combination with the current standard of care (PEG-IFN-α plus ribavirin) in HCV patients to achieve enhanced viral load suppression without reducing the efficacy of any of individual drugs. Combinations of Compound 3 with non-nucleoside (Compound 1) NS5B polymerase inhibitors resulted in additivity. These results indicate that Compound 3 may a

be suitable for exploring drug combinations comprised of multiple classes of specific HCV inhibitors in patients.

Biological Example 3 Compound 4 Combinations Materials and Methods Anti-HCV Agents

Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 were synthesized by Gilead Sciences (Foster City, Calif.). Puromycin, IFN-α and Ribavirin were purchased from Sigma (St. Louis, Mo.). Calcein AM was purchased from Anaspec (Fremo

Calif.).

Cell Line and Cell Culture

The HCV genotype 1a replicon cell line used in this study was described previously. The

were grown in cell culture medium containing Dulbecco's Modified Eagle Medium (DMEM) GlutaMAX (Gibco, Carlsbad, Calif., Cat# 10569-044), supplemented with 10% FBS (HyClone

Logan, Utah, Cat#SH30071.03), 100 Units/mL Penicillin, 100 μg/ml Streptomycin (Gibco, Carlsbad, Calif., Cat# 15140-122), and 0.1 mM non-essential amino acids (Gibco, Carlsbad, Calif., Cat#11140-050). Replicon cells were maintained in 0.5 mg/mL. Geneticin (Invitrogen, Carlsbad, Calif., Cat# 10131-035) to prevent the loss of HCV replicon. The cells were passaged every

days before reaching confluency.

HCV Replicon Assay for EC₅₀, CC₅₀ Determinations and Combination Studies

All compounds were supplied in 100% DMSO except for IFN-α, which was supplied in buff

specified by the manufacture (Sigma, St. Louis, Mo., Cat#14276). Compound serial dilutions were performed in 100% DMSO except for IFN-α, which was serially diluted in cell culture medium described in section 3.2. All serial dilutions were performed in 384-well polypropylene plates (Thermo Scientific, Hudson, N.H., Cat#4341) using a Biomek FX Workstation. For EC₅₀ and CC₅₀ determinations, test compounds were serially diluted in ten steps of 1:3 dilutions

columns 3-20 of the 384-well plates. For combinational studies, one compound was serially diluted in nine steps of 1:2 dilutions toward the horizontal direction wth the other compounc senaiiy diluted in seven steps of 1:2 dilutions toward the vertical direction. This achieved a defined set of different drug concentrations and ratios. For each individual drug, the EC₅₀

was selected as the midpoint for the concentration range tested. All serial dilutions were performed in four replicates per compound within the same 384-well plate, 100% DMSO was added into column 1-2 of each serial dilution 384-well plate. A HCV protease inhibitor ITMN at 100 μM was added into column 23 as a control of 100% inhibition of HCV replication while puremycin at 10 mM was added into column 24 as a control of 100% cytotoxicity.

To each well of a black polystyrene 384-well plate (Greiner Bio-one, Monroe, N.C., Cat#7810 cell culture treated), 90 μL of cell culture medium (without geneticin) containing 2000 suspended HCV replicon cells was added with a Biotek μFlow Workstation. For compound transfer into cell culture plates, 0.4 μL of compound solution from the compound serial dilution plate was transferred to the cell culture plate on a Biomek FX Workstation. The DMSO concentration in the final assay wells was 0.44%. The plates were incubated for 3 days at 37° C. with 5% CO₂ and 85% humidity.

The HCV replicon assay was a multiplex assay which can assess both cytotoxicity and anti-replicon activity from the same well. To CC₅₀ assay was performed first. The media in the 384-well cell culture plate was aspirated and the wells were washed four times with 100 μL 1×PBS each, using a Biotek ELX405 plate washer. A volume of 50 μL of a solution containing 400 nM calcein AM (Anaspec, Fremont, Calif., Cat#25200-056) in 1×PBS was added to each well of the plate with a Biotek μFlow Workstation. The plate was incubated for 30 minutes at room temperature before the fluorescence signal (excitation 490 nm, emission 520 nm) was measured with a Perkin Elmer Envision Plate Reader.

EC₅₀ assay was performed in the same wells as CC₅₀ assay. The calcein-PBS solution in the 384-well cell culture plate was aspirated with a Biotek ELX405 plate washer. A volume of 20 μL of Dual-Glo luciferase buffer (Promega, Madison, Wis., Cat#E298B) was added to eash well of the plate with a Biotek μFlow Workstation. The plate was incubated for 10 minutes at room temperature. A volume of 20 μL of a solution containing 1:100 mixture of Duel-Glo Stop & Glo substrate (Promega, Madison, Wis., Cat#E313B) and Dual-Glo Stop & Glo buffer (Promega, Madison, Wis., Cat#E314B) was then added to each well of the plate with a Biotek μFlow Workstation. The plate was then incubated at room temperature for 10 minutes before the luminescence signal was measured with a Perkin Elmer Envision Plate Reader.

Data Analysis

The cytotoxicity effect was determined by calcein AM conversion to fluorescent product. The percent cytotoxicity was calculated by equation 1;

$\begin{matrix} {{\% \mspace{14mu} {cytotoxicity}\mspace{14mu} {or}\mspace{14mu} \% \mspace{14mu} {inhibition}} = {100 \times \left( {1 - \frac{X_{C} - M_{B}}{M_{D} - M_{B}}} \right)}} & (1) \end{matrix}$

where X_(C) is the fluorescence signal from the compound-treated well; M_(S) is the average fluorescence signal from puromycin-treated wells; M_(D) is the average fluorescence signal from DMSO-treated wells. The anti-HCV replication activity was determined by the luminescence signal generated from the reporter renilia luciferase of the HCV replicon. The percent inhibition on HCV replicon was calculated using equation 1, where X_(C) is the luminescence signal from compound-treated well; M_(S) is average luminescence signal from the ITMN-191-treated wells; M_(D) is the average luminescence signal from DMSO-treated wells.

The CC₅₀ values were determined as the testing compound concentration fast caused a 50% decrease of salt viability. The EC₅₀ values were determined as the testing compound concentration that caused a 50% decrease in HCV replication. Both CC₅₀ and EC₅₀ values were obtained using Pipeline Pilot 5.0 software package (Acceirys, San Diego, Calif.) by nonlinear regression fitting of experimental data to equation 2:

$\begin{matrix} {y = {d + \frac{a - d}{\left\lbrack {1 + \left( \frac{x}{c} \right)^{b}} \right\rbrack}}} & (2) \end{matrix}$

where y is the observed % inhibition of HCV replicon at x concentration of compound; d is estimated response at zero compound concentration; a is estimated response at infinite compound concentration; c is the mid-range concentration (CC₅₀ or EC50); b is the Hill slope factor.

The combination study experimental data were analyzed using the MacSynergy II program developed by Prichard and Shipman. The software (MacSynergy™ II, University of Michigan, Mich.) calculates theoretical inhibition assuming an additive interaction between drugs (based on the Bliss Independence model) and quantifies statistically significant differences between the theoretical and observed inhibition values. Plotting these differences in three dimensions results in a surface where elevatione in the Z-plane represent antiviral synergy and depressions represent antiviral antagonism between compounds. The calculated volumes of surface deviations are expressed in nM²%. Per Prichard and Shipman, combination effects are defined

-   -   Strong synergy: >100 nM²%     -   Moderate synergy: >50 and ≦100 nM²%     -   Minor synergy: >25 and ≦50 nM²%     -   Additivity: ≦25 and >−25 nM²%     -   Minor antagonism: ≦−25 and >−50 nM²%     -   Moderate antagonism: ≦−50 and >−100 nM²%     -   Strong antagonism: ≦−100 nM²%

For each combination study, three independent experiments were performed with four replicates in each experiment.

Results Antiviral Activity and Cytotoxicity of Individual Compounds in HCV Genotype 1a Replicon Assay

The anti-HCV activity and cytotoxicity of Compound 4 and other compounds were tested in Huh-7 cells carrying a HCV genotype 1a raptors. The EC₅₀ and CC₅₀ values are listed in Table VI. There is no significant cytotoxicity observed for aii compounds up to the highest concentrations tested.

TABLE VI EC₅₀ and CC₅₀ of Compounds used in this Study against HCV Genotype 1a Replicon Compounds EC₅₀ ^(a) (nM) CC₅₀ ^(a) (nM) Compound 1 19 ± 8  >44400 Compound 2 496 ± 135 >22200 Compound 3 49 ± 18 >22200 Compound 4 201 ± 74  >44400 Compound 5  15 ± 2.4 >44400 Compound 6 0.033 ± 0.011 >44400 IFN-α  1.4 ± 0.3^(b)    >50^(b) Ribavirin 36482 ± 17507 >88800 ^(a)Values are average ± standard deviation for three or more independent experiments ^(b)IFN-α values are expressed in Units (U) per milliliter (mL) instead of a nanomolar concentration

Antiviral Activity and Cytotoxicity of Compound 4 in Combination with Other Classes of Anti-HCV Agents

The antiviral of effects of Compound 4 in combination with other anti-HCV compounds were evaluated using the HCV genotype 1a replicon. The results were analyzed using MacSynergy II, which provides surface plots displaying significant deviations from additivity. Synergy and antagonism volumes (nM²%) calculated from deviations from additive surface are summarized in Table VII. At 95% confidence interval, the mean synergy and antagonism volumes are between 25 and −25 nM² % when Compound 4 was combined with IFN-α, Compound 2 and Compound 6, indicative of additive interaction with those compounds. Furthermore, Compound 4 shows synergy volumes in the range of 25 to 50 nM²% when combined with Compound 1, Compound 5 or Compound 3, suggesting miner synergistic interaction.

TABLE VII Quantification of Antiviral Synergy and Antagonism and Drug Interactions for Drug Combinations with Compound 4 Drug(s) Used in Antagonism Combination Synergy Volume Volume with Compound 4 (nM² %)^(a) (nM² %)^(a) Interaction Compound 1 34 ± 26 −1 ± 2 Minor synergy Compound 2 22 ± 14 −2 ± 3 Additivity Compound 3 26 ± 6  −3 ± 2 Minor synergy Compound 5 26 ± 28 −1 ± 3 Minor synergy Compound 6 19 ± 17 −7 ± 7 Additivity IFN-α 12 ± 6   0 ± 0 Additivity Ribavirin 1 ± 1 −43 ± 20 Minor antagonism Values represent the mean ± standard deviation of three independent experiments performed in four replicates

In all combination studies, the cell viability is higher than 85% at all concentration ratios and all drug combinations show additive effects on the cytotoxicity as shown in Table VIII.

TABLE VIII Quantification of Cytotoxicity Synergy and Antagonism and Drug Interactions for Drug Combinations with Compound 4 Drug(s) Used in Combination Synergy Volume Antagonism with Compound 4 (nM² %)^(a) Volume (nM² %)^(a) Interaction Compound 1 13 ± 11 0 ± 1 Additivity Compound 2 17 ± 14 0 ± 0 Additivity Compound 3 3 ± 5 0 ± 0 Additivity Compound 5 15 ± 8  −10 ± 7  Additivity Compound 6 8 ± 4 0 ± 0 Additivity IFN-α  8 ± 12 −7 ± 13 Additivity Ribavirin 4 ± 3 −1 ± 2  Additivity ^(a)Values represent the mean ± standard deviation of three independent experiments performed in four replicates However, Compound 4 shows an antagonism volume of −43 nM²% when combined with Ribavirin, suggesting a minor antagonistic interaction.

TABLE IX Quantification of Cytotoxicity Synergy and Antagonism and Drug Interactions for Drug Combinations with Ribavirin Drug Used in Combination Synergy Volume Antagonism with Ribavirin (μM² %)^(a) Volume (μM² %)^(a) Interaction Compound 4 4 ± 3 −1 ± 2 Additivity ^(a)Values represent the mean ± standard deviation of three independent experiments performed in four replicates

The Ribavirin concentration that shows the highest antagonism with Compound 4 is around 0.5 to 1 μM, which is about 10-fold lower than the steady-state plasma concentration of Ribavirin (6-11 μM) observed in human at a dose of 800 mg/day. At this physiological concentration of Ribavirin (6-11 μM), the antagonistic interaction between Ribavirin and Compound 4 is minimal across a wide range of Compound 4 concentrations (0-0.44 μM). Therefore, the observed minor antagonism between Ribavirin and Compound 4 in the in vitw replicon system is unlikely to have clinical significance.

Conclusions

The antiviral activity of Compound 4 (in a diastereomeric mixture) was tested in combination with the current standard of care (IFN-α/Ribavirin), as well as Gilead Sciences’ internal developmental candidates Compound 1 and Compound 5 (non-nucleosida NS5B inhibitors), Compound 2 and Compound 3 (NS3 protease inhibitors), and Compound 6 (NS5A inhibitor). As summarized in Table VIII, Compound 4 showed additive antiviral activity in combination with IFN-α, Compound 2 and Compound 6 and minor synergy with Compound 1, Compound 5 and Compound 3.

The combination of Compound 4 with Ribavirin resulted in a minor antagonism at Ribavirin concentrations between 0.5 to 1 μM, which is approximately 10-fold lower than its steady-state physiological concentration (6-11 μM) in human plasma. At the clinically relevant Ribavirin concentration, the antagonistic interaction between the two compounds became negligible.

Biological Example 4 Compound 5 Combinations

The antiviral activity of Compound 5 was tested in GT-1b Huh-lunet cells (using substantially the same methods as in the assays for Compound 4) in combination with the internal developmental compounds Compound 1, Compound 2 and Compound 3 (NS3protease inhibitors), Compound 5 (NS5A Inhibitor), Compound 4 (C-nuc NS5B inhibitor) and also the approved HCV merapeuties PEG-IFN-α and Ribavirin. Combination data were analyzed based on the Bliss independence model using MacSynergy II software. Results of the combination assays ware expressed as mean synergy and antagonism volumes (nM²) calculated at 95% confidence from two independent experiments performed in triplicate. Combination effects are defined as:

-   -   Strong synergy if volumes>100 nM²; this amount of synergy is         probably important in vivo     -   Moderate synergy if volumes are >50 and ≦100 nM²; this amount of         synergy may be important in vivo     -   Minor synergy if volumes are >25 and <50 nM²     -   Additivity if volumes are >−25 and ≦25 nM²     -   Minor antagonism if volumes are <−25 and >−50 nM²     -   Moderate antagonism if volumes are >−100 nM² and ≦−50 nM²; this         amount of antagonism may be important in vivo     -   Strong antagonism if volumes are ≦−100 nM²; this amount of         antagonism is probably important in vivo.

The combination of the allosteric NS5B inhibitors Compound 1 and Compound 5 resulted in moderate synergy in the replicon assay. Studies with other HCV inhibitors, including PEG-IFN-α and Ribavirin, revealed additive to minor synergistic interactions.

TABLE X Antiviral effects of Compound 5 in combination with other anti-HCV drugs in 1b Huh-luc replicon cells Drug used in combination Synergy Volume Antagonism with Compound 5 (nM²)^(a) Volume (nM²)^(a) Interaction Compound 1 70 ± 26  0 ± 0 Moderate synergy Compound 2 22 ± 12 −7 ± 7 Additive Compound 3 19 ± 13 −2 ± 2 Additive Compound 4 26 ± 28 −1 ± 3 Minor synergy Compound 6 34 ± 19  0 ± 0 Minor synergy PEG-IFN-α 31 ± 23 −2 ± 4 Minor synergy Ribavirin 12 ± 8  −12 ± 9  Additive ^(a)Values represent the mean ± standard deviation of two independent experiments performed in triplicate

Biological Example 5 Compound 6, Combinations Materials and Methods Compounds

Compound 1, Compound 2, Compound 3, Compound 6 and Compound 7 wars synthesized by Gilead Sciences (Foster City, Calif.), IFN-α and Ribavirin were purchased from Sigma (St Louis, Mo.).

Cell Lines

HCV genotype 1b replicon cells (Huh-luc) were obtained from Reblikon (Mainz, Germany). The replicon in these cells is designated I389luc-ubi-neo/NS3′/ET and encodes a selectable resistance marker (neomycin phosphotransferase) as well as the firefly luciferase reporter gene. Huh-luc cells were maintained in Dulbecco's Modified Eagle's Medium GlutaMax (DMEM; Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah), 1× penicillin/streptomycin, 1× nonessential amino acids and 0.5 mg/mL of G-418 (all from Invitrogen, Carisbad, Calif.). Cells were passaged twice a week and maintained at subconfluent levels.

Assays Antiviral Activity Assay in HCV Huh-luc Replicon Cells

Replicon cells were seeded in 96-well plates at a density of 7×10³ cells per well in 100 μL of DMEM culture medium, excluding G-418. Compounds were serially diluted 1:2 in 100% DMSO. Serial dilutions were added to the cells at a 1:200 dilution to achieve a final concentration of 0.5% DMSO in a total volume of 200 μL. Rates were incubated at 37° C. for 3 days, after which culture media were removed and cells were lysed and assayed for luciferase activity using a commercial luciferase assay (Fromega, Madison, Wis.).

Antiviral Combination Studies

Replicon cells were seeded in 90-well plates at a density of 7×103 cells per well in 100 μL culture medium, excluding G-418. Compound 8 and other compounds were serially diluted 1:2 in 100% DMSO and added in a matrix format to 96-well plates, achieving a defined set of different drug concentrations and ratios in a final volume of 200 μL and a final DMSO concentration of 0.5%. For each individual drug, the EC₅₀ value was selected as the midpoint for the concentration range tested. Cells were incubated for 3 days and analyzed for luciferase expression using a commercial luciferase assay (Fromega). For each combination study, two independent experiments were performed in triplicate.

Cellular Cytotoxicity Determination

Replicon cells were seeded and treated with drugs as described for the antiviral combination studies above. After three day incubation at 37° C., the culture media was removed and cells were lysad and assayed for cytotoxicity using a CellTiter-Gio Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. Relative Light Units were converted into percentages relative to the untreated controls (defined as 100%).

Data Analysis EC₅₀ Calculations

Following EC₅₀ assays, luclferasa levels in drug-treated samples were expressed as a percentage of those in untreated controls (defined as 100%). EC₅₀ values were calculated by nonlinear regression analysis of replicate data sets using XLfit 4 software (IDBS, Emeryville, Calif.).

Calculation of Antiviral Synergy and Antagonism

Following combination assays, luciferase levels in drug-treated samples were expressed as a percentage of those in untreated controls (defined as 100%), Replicate data sets were then analyzed using the fvlaeSynergy II program developed by Prichard and Shipman. The software (MacSynergy™ II, University of Michigan, Mich.) calculates theoretical inhibition assuming an additive interaction between drugs (based on the Bliss independence model) and quantifies statistically significant differences between the theoretical and observed inhibition values. Plotting these differences in three dimensions results in a surface where elevations in the Z-plane represent antiviral synergy and depressions represent antiviral antagonism between compounds. The calculated volumes of surface deviations are expressed in nM²%. Per Prichard and Shipman, combination effects are defined as:

-   -   Strong synergy if volumes>100 nM²; this amount of synergy is         probably important in vivo     -   Moderate synergy if volumes are >50 and ≦100 nM²; this amount of         synergy may be important in vivo     -   Minor synergy if volumes are >25 and <50 nM²     -   Additivity if volumes are >−25 nM2 and ≦25 nM²     -   Minor antagonism if volumes are <−25 and >−50 nM²     -   Moderate antagonism if volumes are >−100 nM² and ≦−50 nM² this         amount of antagonism may be important in vivo     -   Strong antagonism if volumes are ≦−100 nM²; this amount of         antagonism is probably important in vivo.

Results Antiviral Activity of Individual Compounds in Huh-luc Replicon Cells

Prior to initiating combination experiments, the antiviral activity of individual compounds was determined in Huh-luc replicon cells. EC₅₀ values consistent with historical results were observed with all seven compounds.

TABLE XI Individual EC₅₀ Values for Anti-HCV Compounds in Huh-luc Replicon Cells Compound EC₅₀ (nM)^(a) IFN-α^(b) 0.05 U/ml ± 0.04    Ribavirin >12 ± 2.4  Compound 1 0.96 ± 0.39 Compound 2 5.0 ± 0.0 Compound 3 3.0 ± 1.2 Compound 6 0.0018 ± 0.0007 Compound 7 1245 ± 341  ^(a)EC₅₀ indicates arithmetic mean ± standard deviation for three or more independent experiments. ^(b)IFN-α EC₅₀ is expressed in Units (U) per milliliter (mL) instead of a nanomolar concentration.

Combination Antiviral Effects and Drug Interactions

The antiviral effects of Compound 6 in combination with other HCV inhibitors were evaluated using the HCV 1b replicon system. The resulting data were analyied using MacSynergy II, which provides surface plots displaying significant deviations from additivity. Quantification of statistically significant deviations from additivffy from two independent experiments is summarised in Table XII. Combinations of Compound 6 with IFN-α or Compound 1 resulted in synergy volumes of 32 and 34 nM², respectively, indicating minor synergy. Ribavirin, Compound 2 and Compound 7 yielded synergy volumes of 61, 52 and 51 when combined with Compound 6, respestively, indicating a moderate synergistic interaction between Compound 6 and these three HCV inhibitors. The combination of Compound 6 with Compound 3 resulted in a synergy volume of 132 nM²% signifying a strongly synergistic antiviral interaction. None of the compounds yielded antiviral antagonism volumes outside of the additive range (>−25 nM) when combined with Compound 6.

TABLE XII Quantification of Antiviral Synergy and Antagonism and Drug Interactions for Drug Combinations with Compound 6 Drug(s) Used in Synergy Combination Volume Antagonism with Compound 6 (nM²)^(a) Volume (nM²)^(a) Interaction IFN-α 32 ± 1.4  0.0 ± 0.0 Minor Synergy Ribavirin 61 ± 0.5 −0.5 ± 0.1 Moderate Synergy Compound 1 34 ± 9.9  −17 ± 0.7 Minor Synergy Compound 2 52 ± 5.1 −0.7 ± 0.7 Moderate Synergy Compound 3 132 ± 44   −0.1 ± 0.2 Strong Synergy Compound 7 51 ± 7.8 −0.2 ± 0.1 Moderate Synergy ^(a)Values represent the arithmetic mean ± standard deviation of two independent experiments performed in triplicate.

Cell Viability Percentages for Compound 6 in Combination with Other HCV Inhibitors

To ensure that antiviral combination results were not confounded by combination cytotoxicity, the cytotoxicity was investigated in parallel using the same compound concentrations tested in the antiviral assays (Table XIII). For ail compounds, cell viability was at least 98% of untreated controls for combinations at the highest concentrations tested. Therefore, no significant in wfro cytotoxicity was observed while testing Compound 6 alone, or in combination with these agents.

TABLE XIII Cell Viability Percentages for Compound 6, Combinations in Huh-luc Replicon Cells Compounds Concentration(s) (nM) Cell Viability %^(a) Compound 6 0.014  99 ± 1 Compound 6 + IFN-α^(b) 0.014 + 0.8 102 ± 3 Compound 6 + Ribavirin 0.014 + 8000 105 ± 4 Compound 6 + Compound 1 0.014 + 4.0  99 ± 3 Compound 6 + Compound 2 0.014 + 24.0 103 ± 3 Compound 6 + Compound 3 0.014 + 12.8 104 ± 4 Compound 6 + Compound 7 0.014 + 8800 103 ± 3 ^(a)Cell viability % indicates arithmetic mean ± standard deviation for at least two independent experiments performed in triplicate. ^(b)IFN-α is expressed in Units (U) per milliliter (mL) instead of a nanomolar concentration.

Conclusions

Results of these in vitro experiments indicate that Compound 6 has minor antiviral synergy when combined with IFN-α or the non-nucleoside NS5B polymerase inhibitor Compound 1. Combinations of Compound 6 with Ribavirin, the NS3 protease inhibitor Compound 2 or the nucleoside NS5B polymerase inhibitor Compound 7 resulted in moderate antiviral synergy. Strong antiviral synergy was observed between Compound 6 and the NS3 protease inhibitor Compound 3. No significant in vitro cytotoxicity was identified while testing these drug combinations. These rssuits suggest that Compound 6, Could rationally be combined with the current standard of care.

Biological Example 6 Compounds

Compound 1, Compound 3, Compound 4, and Compound 6 were Synthesized by Gilead Sciences (Foster City, Calif.)

Cell Lines

HCV genotype 1b replicon cells (Huh-luc) were obtained from Reblikon (Mainz, Germany). The replicon in these cells is designated 1389luc-ubi-neo/NS3-3′/ET and encodes a selectable resistance marker (neomycin phosphotransferase) as well as the firefly lusiferase reporter gene. Huh-luc cells ware maintained in Dulbecco's Modified Eagle's Medium GlutaMax (DMEM; Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah), 1× penicillin/streptomycin, 1× nonessential amine adds and 0.5 mg/mL of G-416 (all from Invitrogen, Carlsbad, Calif.). Cells were passaged twice a week and maintained at subconfluent levels.

Assays

Determination of Compound Concentration Required to Suppress Replicon RNA by 1-1.5 Log Over 6 Days of Treatment

Genotype 1b replicon cells were seeded in T-75 flasks at a cell density of 2.5×10⁵ cells/flask, excluding G418. Compounds were individually added to the cells at variable concentrations: Compound 6 was added at concentrations of either 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, or 12 μM, Compound 4 was added at 125 nM, 250 nM, 375 nM, 500 nM or 1000 nM, Compound 1 was added at 1.25 nM, 2.5 nM, 5 nM, 2.75 nM or 10 nM, and Compound 3 was added at concentrations of 3.75 nM, 7.5 nM, 11.25 nM, 15 nM, 30 nM or 60 nM. Flasks were incubated at 37° C., media and compounds were refreshed every two days. After 6 days of incubation the replicon cells were collected for RNA enaction and replicon RNA QRT-PCR analysis.

Compound Combination Replicon Cure Assay

Genotype 1b replicon cells were seeded in T-75 flasks at a density of 2.5×10⁵ cells/flask Compounds were added to the T-75 flasks at the following concentrations; Compound 6 at 4 μM, Compound 4 at 1000 nM, Compound 1 at 10 nM, and Compound 3 at 26.25 nM. Flasks ware incubated at 37° C. and compounds and media were refreshed every two days. All experiments were performed in duplicate and will be noted in as flask 1 and flask 2. On day 6 all cells were eelleded form flask 1 for RNA extraction followed by HCV replicon specific QRT-PCR analysis and the cells form flask 2 were replated on a 10 cm tissue culture dishes in the presence of G418 for 14 days to record colony formation of uncured replicon cells.

QRT-PCR Assay

Total RNA was extracted with the RiboPure kit (AM1924 Life Technologies Corporation Carlsbad, Calif.) following the manufacturer's protocol. Extracted RNA samples were stored at −80° C. until use. For the Quantitative RT-PCR assay the Qiagan One-step QRT-PCR kit was used according to manufacturer's protocol (Qiagan, Valencia Calif.). The genotype 1b HCV NS3 gene specific primers, forward primer NS3_(—)160FL 5′-CGGCGGACTGTGTATCATGGTGC[FAM]G-′3 (SEQ ID NO:1) and reverse NS3_(—)180 5′-GGTCCTGGTCCACATTGGTGT-′3 (SEQ ID NO:2) and 18S rRNA LUX™ [FAM] endogenous control primers set (115HM-01) were produced by invitrogan corporation (Carlsbad, Calif.). For the reverse transcriptase step, the reactions were incubated at 44° C. for 30 mm, the reverse transcriptase enzyme was them degraded by heating the sample to 94° C. for 10 min. The Q-PCR step included 38 cycles at 84° C. for 15 s and 58° C. for 30 s.

Results

Prior to initiating combination replicon cure expehments the compound concentration required fo suppress genotype 1b repilcen RNA by 1-1.5 log was determined for Compound 6, Compound 4, Compound 1, and Compound 3. The replicon RNA log drop is relative to the RNA levels in DMSO control treated replicon cells maintained for 6 days.

TABLE XIV Individual compound dose able to induce replicon RNA 1-1.5 log drop in a 6 day assay Compound concentration Compound Replicon RNA log drop (nM) Compound 1 −1.0 10 Compound 3 −0.9 26.25 Compound 4 −1.2 1000 Compound 6 −1.4 0.004

Combination Genotype 1b Replicon Cure Assay

The replicon RNA suppression by compounds Compound 6, Compound 4, Compound 1 and Compound 3 was determined in a 6 day assay as individual compounds and in various double, triple, and quadruple combinations. The replicon RNA log drop is relative fo the RNA levels in DMSO control treated replicon cells maintained for 6 days alongside the treatment flasks. The ability of the various compound combinations to cure the cells from the HCV replicon was determined by colony formation. Colony formation occurred after compound treatment was removed and G418 pressure was returned for 14 days. If a compound combinaion completely cures the cell population from the HCV replicon no colonies will develop since the cells lack resistance to G418.

TABLE XV Quantification of compound combination in the replicon cure assay Replicon Uncured colony Compounds RNA log drop number Compound 6 −1.4 634 Compound 4 −1.2 1054 Compound 1 −1.0 657 Compound 3 −0.9 989 Compound 4 + Compound 6 −2.67 15 Compound 1 + Compound 4 −2.022 14 Compound 3 + Compound 4 −2.26 23 Compound 1 + Compound 6 −2.3 148 Compound 3 + Compound 6 −2.62 13 Compound 1 + Compound 3 −1.8 113 Compound 1 + Compound 4 + −2.66 0 Compound 6 Compound 3 + Compound 4 + −2.71 0 Compound 6 Compound 1 + Compound 3 + −2.69 0 Compound 4 Compound 1 + Compound 3 + −2.69 0 Compound 6 Compound 1 + Compound 3 + −2.71 0 Compound 4 + Compound 6 DMSO (0.2% to match 0 6330 Quadruple combination)

Conclusions

Results of these in vitro experiments indicate that combination of two compounds increases the viral RNA tog drop over 6 day treatment and increases the rate of cured replicon cells. The dual combinations of Compound 6 with Compound 4 or Compound 3 results in larger replicon RNA log supression and lowest number of uncured colonies compared to all other dual compound combinations. The combination of three or four compounds cures all replicon cells and the combination treatments suppress the replicon RNA levels to the assay limit of detection.

Biological Example 7 Compound 10 and Compound 5 Cross-Resistance

This study was conducted to determine the in vitro cross-resistance profiles of Compound 10 and Compound 6. A panel of HCV mutant repiieens bearing the signature NS5B nucleoside HCV drug resistance mutation S282T or the most common in vitro and in vivo NS5A HCV drug resistance mutations was investigated via transient replicon assay to determine whether cross-resistance exists between mutations conferring reduced susceptibility to Compound 10 or Compound 6. No cross-resistance was observed between these compound with S282T mutant replicons remaining fully susceptible to Compound 6 and NS5A mutants showing no reduced susceptibility to Compound 10.

Materials and Methods Reagents Compounds

Compound 8 and Compound 10 were syrthesteed st Gilead Sciences, Inc. in Foster City, Calif.

Cell Lines

Huh-lunet, a Huh-7 clone that is highly permissive for HCV replication, was obtained from ReBLikon GmbH (Mainz, Germany). Huh-lunet cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; GIBCO, Carisbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Hyclene, Logan, Utah). Cells were maintained at 37° C. in humidified incubators (85% humidity) and under a 5% CO₂ atmosphere.

PI-hRluc, a bicistronic replicon encoding the Renilla luciferase gene downstream of the polio IRES and the genotype 1b (Con-1 strain) HCV nonstructural genes (NS3-NS5B) downstream of the EMCV IRES, was used for transient transfecfion studies. The plasmid pPI-Rluc was generated from the plasmid pFKI341 PI-Luc/NS3-3′/ET, which encodes a genotype 1b (Con-1 strain) subgenomic replicon and was obtained from ReBLikon (Friebe et al., J Virol 2001;75 (24):12047-57). The hRluc gene was PGR amplified from pF8 CMV hRluc-neo Flexi(R) (Promega, Madison, Wis.) by PCR using Accuprime Super Mix I (Invitrogen, Carlsbad, Calif.) and the primers PV_Rluc_Top and 3′-Rluc(NotI). These two primers have the following sequences and carry restriction sites for subsequent cloning: PV_Rluc_Top: 5′ ATC AGA CAA TTG TAT CAT AAT GGC TTC CAA GGT GTA CG 3′ (SEQ ID NO:3); 3′-Rluc(NotI): 5′ ACG TCA CTA TCT ACG CGG CCG CTT ACT GCT CGT TCT TC3′ (NotI site underlined) (SEQ ID NO:4). The T7 Promoter, 5′UTR and Polio Virus IRES were PCR emptied from the plasmid pFKI341 PI-Luc/NS3-3/ET using the primers 5′-P7(SbfI) and PV_Rluc_Bottom. These two primers have the following sequences and carry restriction sites for subsequent cloning; 5′-P7(SbfI): 5′ CAA GCT AAG CTG CCT GCA GG T 3′ (SbfI site underlined) (SEQ ID NO:5); PV_Rluc_Bottom: 5′ CGT ACA OCT TGG AAO CCA TTA TGA TAC AAT TGT CTG AT (SEQ ID NotI). The subsequent PCR fragments from the two above reactions were then joined together using overlapping PCR and the primers 5′-P7(SbfI) and 3′-Rluc(NotI). The completed P7-5′UTR-Polio Virus IRES-hRluc amplification product was subcloned into pCR2.1-TOPO. The resulting plasmid was digested with SbfI and NotI, and the excised fragment (P7-5′UTR-Polio Virus IRES-hRluc) were ligated using T4 DNA ligase into pFKI341 PI-Luc/NS3-3′/ET digested wth the same enzymes. The resulting vector, pPI-hRluc, was sequenced to confirm the correct orientation and sequence of the P75′UTR-Polio Virus IRES-hRluc region of the plasmid.

GT 1a and 2a replicens containing hRluc have been described previously (Robinson M, Yang H, Sun S C, Peng B, Tian Y, Pagratis N, at al. Novel HCV Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a. Antimicrob Agents Chemother 2010.)

The PI-hRluc replicon was used as a backbone for chimera abstraction. An internel deletion was made in NS5B rendering it replication deficient. The last 413 base pairs (encoding last 137 NS5A amino acids) of 1b-con-1 NS5A were synthesized in frame with NS5B sequences from genotypes 2b, 3a, 4a, 5a, and 6a (Genscript Inc. Piscataway N.J.). Consensus NS5B sequences for each of these genotypes were derived by aligning sequences deposited in the European HCV database and extracting a consensus. These novel consensus sequences for NS5B, as well as sequences derived from individual clinical isolates (Hertihy et al., Antimicrob Agents Chemother 2008;52 (10):3523-31) were used to construct the NS5B chimeric replicons. A unique XhoI site was used at the 5′ and and a unique AseI site at the 3′ end to directionally clone into the 1b-hRLuc/NeoR NS5B vector via standard molecular biology techniques.

NS5B S282T mutations were introduced replicon plasmids using the QuikChange II XL mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolia, Calif.). The entire mutated replicon was sequenced fo confirm the presence of the S282T mutation and absence of any secondary site mutations.

NS5A mutant replicons were created by synthesizing a sequence encoding the first 149amino acids of containing the desired mutation flanked by a 5′ and 3′ BsrGI and EcoRI site, respectively (Genscnpt, Piscataway, N.J.). Synthesized fragments were then cloned by standard molecular biology techniques info a 1a Rluc replicon plasmid modified to enable cloning info unique BsrGI and EcoRI restriction sites. Mutations were confirmed by DNA sequencing.

Replicons were linearised using the following enzymes: Spe I (1b PI-Rluc-based replicons), Hpa I (1a-Rluc-based replicons), and XbaI/HpaI (2a Rluc replicon). Replicon RNAs were transcribed in vitro from replicon-encoding plasmids using a T7 Ribomax in vitro transcription kit (Promega; Madison, Wis.) followed by purification using an RNAeasy column (Qiagen; Valencia, Calif.).

Assays Drug Susceptibility Determination using Transient Transfection Replicon Assay

RNA was transfected into Huh-lunet cells using the method of Lohmann et al. (Lohmann et al., Science 1999;285 (5424):110-3) Briefly, cells were trypsinized and washed twice with PBS. A suspension of 4×10⁸ cells in 400 μL of PBS were mixed with 5 μg of RNA and subjected to eiectroperetlon using settings of 960 pF and 270 V. Cells were transferred into 40 mL of pre-warmed culture medium and then seeded into 96-well or 384-well plates. Compounds were 3-fold serially diluted in 100% DMSO and added to cells to achieve a final DMSO concentration of 0.5%. Cells were treated for three days after which culture media were removed, cells were lysed, and Renilla luciferase activity was quantified using commercially available reagents (Promega) and a Victor or Envision instrument (Perkln Elmer, Waltham, Mass.).

Data Analysis

Date were converted irrte percentages relative ts untreated controls (defined as 100%), and EC₅₀ values were calculated by non-linear regression of two replicate data sets using GraphPad Prism software or Pipeline Pilot Resistance fold changes were catenated as the ratio of mutant to wild-type replicon.

Results Activity of Compound 10 and Compound 6 against S282T

Previous in vitro resistance selection with Compound 10 has consistently identied S282T in NS5B as primary mutation in multiple genotypes (GT 1b, 1a and 2a). The NS5B S282T mutation was subsequently introduced into the full-length GT 1a, 1b, and 2a repiicons and chimeric repiicons containing a 2b, 3a, or 4a MSS8 sequence cloned into a GT1b backbone. The susceptibility to Compound 10, Compound 6, and ribavirin (RBV) was evaluated using a transient replication assay. The S282T mutation displayed a reduced susceptibility to Compound 10 with EC₅₀ values across all five genotypes ranging from 117.1 nM to 346.1 nM. The fold increase in EC₅₀ for S282T ranged from 2.4 to 16.0 compared to the wild-type from the corresponding genotypes demonstrating decreased replicon susceptibility to Compound 10 when the NS5B S282T mutation is present.

For the wild-type replicon, the EC₅₀ values for RBV were also similar across the five genotypes tested with the lowest ECgbeing against GT 2b. Interestingly, the EC₅₀ values for S282T replicons were 5- to 10-fold more sensitive to treatment with RBV than their corresponding wild-type for all five genotypes. No significant differences were observed in Compound 6 EC₅₀ between the wild-type and S282T repfeons indicating that this mutation does not alter susceptibility to Compound 6, in conclusion, while the S282T replicon conferred reduced susceptibility to Compound 10, the mutant replicon demonstrated wild-type sensitivity to Compound 6 and an increased susceptibility to inhibition by RBV over wild-type,

TABLE 7.1 Antiviral activity of Compound 10 and RBV against wild-type and S282T mutant of GT1b, 2a, 2b, 3a and 4a Compound 10 RBV Compound 6 EC₅₀ nM^(a) Fold EC₅₀ nM^(a) Fold EC₅₀ nM^(a) Fold Replicon WT S282T change^(b) WT S282T change^(b) WT S282T change^(b) GT1b 21.5 189.2 8.8 6.6 1.6 0.2 0.5 0.4 0.8 GT2a 146.8 346.1 2.4 8.3 0.6 0.1 5165 2336 0.5 GT2b 13.3 215.6 16.2 2.6 0.6 0.2 0.5 0.5 0.9 GT3a 33.9 117.1 3.5 6.7 1.0 0.2 0.4 0.2 0.6 GT4a 35.8 217.5 6.1 6.2 0.6 0.1 0.5 0.4 0.6 ^(a)EC₅₀ indicates average for two or more independent experiments. ^(b)Fold change from the corresponding wild type

Activity of Compound 10 and Compound 6 against NS5A Mutants

To determine if NS5A drug resistance mutations are oross resistant to Compound 10, a panel of NS5A mutant replicons was assayed for susceptibility to both Compound 6 and Compound 10. All seven NS5A mutants displayed a reduced susceptibility to Compound 6 with an increase in EC₅₀ ranging from 25- to 3020-fold. In contrast, no significant shift in EC₅₀ was observed for the NS5A mutants to Compound 10 or to a RBV control.

TABLE 7.2 In Vitro Activity of Compound 10 or Compound 6 against NS5A Mutants in Genotype 1a Fold Shift in EC₅₀ (DRM EC₅₀/1a-H77 EC₅₀) Compound M28T Q30H Q30R Q30E L31M Y93C Y93H Compound 25 73 170 997 140 327 3029 6 Compound 0.9 1.0 0.8 1.0 1.1 ND 0.7 10 RBV 0.4 0.7 0.8 0.8 0.5 1.0

Conclusions

In this example, the cross-resistance profiles of Compound 10 and Compound 6 were evaluated using transient HCV repSicone encoding known resistance mutations in NS5A and NS5B conferring reduced susceptibility to Compound 6 and Compound 10, respectively. NS5B S282T replicons conferred reduced susceptibility to Compound 10, while there were no significant differences in Compound 6 EC₅₀ measured from wild-type and S282T replicons. Reciprocally, mutations conferring reduced susceptibility to Compound 6 remained sensitive to treatment with Compound 10.

Overall, these results indicate that resistance mutations for Compound 10 and Compound 8 do not demonstrate cross-resistance and support the use of these compounds in future combination therapy for the treatment of HCV.

Biological Example 8: Combination Activity

Combination study data of Compound 10 with the NS5A inhibitor Compound 6, the non-nucleoside inhibitors Compound 1 or Compound 5, the protease inhibitor Compound 3, or ribavirin (RBV) is shown for an in vitro replicon assay which remains the standard for evaluating the cell-based antiviral activity of HCV inhibitors. These results indicated that Compound 10 has eddive antiviral activity when combined with Compound 6, Compound 1, Compound 5, or Compound 3. In addition, Compound 10 demonstrated minor synergy in combination with RBV in vitro.

Materials and Methods Cell Line and Cell Culture

The HCV genotype 1a replicon cell line used in this study was described previously (Robinson M, Yang H, Sun S C, Peng B, Tian Y. Pagratis N, et al. Novel HCV Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a. Antimiorob Agents Chemother 2010). The cells were grown in cell culture medium containing Dulbecco's Modified Eagle Medium (DMEM) with GlutasMAX (Gibco, Carlsbad, Calif. Cat# 10569-044), supplemented with 10% FBS (HyClona, Logan, Utah, Cat# SH30071.03), 100 Units/mL Penicillin, 100 μg/mL Streptomycin (Gibco, Cadsbad, Calif., Cat# 15140-122), and 0.1 mM non-essential amino adds (Gibco, Cadsbad, Calif., Cat#11140-050), Replicon cells were maintained in 0.5mg/mL Geneticin (Invitrogen, Carlsbad, Calif., Cat# 10131-035) to prevent the loss of HCV replicon. The cells were passaged every 3-4 days before reaching confiuency.

HCV Replicon Assay for EC₅₀, CC₅₀ Determinations and Combination Studies

All compounds were supplied in 100% DMSO. Compound serial dilutions were performed in 100% DMSO. All serial dilutions were performed in 334-well polypropylene plates (Thermo Scientific, Hudson, N.H., Cat#4341) using a Biomek FX Workstation. For EC₅₀ and CC₅₀ determinations, test compounds were serially diluted in fen steps of 1:3 dilutions in columns 3-20 of the 384-well plates. For combinational studies, one compound was serially diluted in nine stepa of 1:2 dilutions toward the horizontal direction with the other compound serially diluted in seven steps of 1:2 dilutions toward the vertical direction. This achieved a defined set of different drug concentrations and ratios. For each individual drug, the EC₅₀ value was selected as the midpoint for the concentration range tested. All serial dilutions were performed in four replicates per compound within the same 384-well plate. 100% DMSO was added into column 1-2 of each serial dilution 384-well plate. A HCV protease inhibitor ITMN-191 at 100 μM was added into column 23 as a control of 100% inhibition of HCV replication white puromycin at 10 mM was added into column 24 as a control of 100% cytotoxicity.

To each well of a black polystyrene 384-well plate (Greiner Bio-one, Monroe, N.C., Cat#781086, cell culture treated), 90 μL of cell culture medium (without geneticin) containing 2000 suspended HCV replicon cells was added with a Biotek μFlow Workstation. For compound transfer into cell culture plates, 0.4 μL of compound solution from the compound serial dilution plate was transferred to the cell culture plate on a Biomek FX Workstation. The DMSO concentration in the final assay wells was 0.44%. The plates were incubated for 3 days at 37° C. with 5% CO₂ and 85% humidity.

The HCV replicon assay was a multiplex assay which can assess both cytotoxicity and anti-replicon activity from the same well. The CC₅₀ essay was performed first. The media in the 384-well cell culture plate was aspirated and the wells were washed four times with 100 μL 1×PBS each, using a Biotek ELX405 plate washer. A volume of 50 μL of a solufjon containing 400 nM caleein AM (Anaspec, Fremont, Calif., Cat#25200-056) in 1×PBS was added to each well of the plate with a Biotek μFlow Workstation. The plate was incubated for 30 minutes at room temperature before the fluorescence signal (excitation 490 nm, emission 520 nm) was measured with a Parkin Elmer Envision Plate Reader.

EC₅₀ assay was performed in the same wells as CC₅₀ assay. The calcein-PBS solution in the 384-well cell culture plate was aspirated with a Biotek ELX405 plate washer. A volume of 20 μL of Dual-Glo luciferase buffer (Promega, Madison, Wis., Cat#E298B) was added to each well of the plate with a Biotek uFiow Workstation. The plate was incubated for 10 minutes at room temperature. A volume of 20 μL of a solution containing T.100 mbdum of Dual-Glo Stop & Glo substrate (Fromega, Madison, Wis., Cat#E313B) and Dual-Glo Stop & Glo buffer (Promega, Madison, Wis., Cat#E314B) was then added to each well of the plate with a Biotek μFlow Workstation. The plate was then incubated at room temperature for 10 minutes before the luminescence signal was measured with a Parkin Elmer Envision Plate Reader.

Data Analysis

The cytotoxicity effect was determined by calcein AM conversion to fluorescent product. The percent cytotoxicity was calculated by equation 1:

$\begin{matrix} {{\% \mspace{14mu} {cytotoxicity}\mspace{14mu} {or}\mspace{14mu} \% \mspace{14mu} {inhibition}} = {100 \times \left( {1 - \frac{X_{C} - M_{B}}{M_{D} - M_{B}}} \right)}} & (1) \end{matrix}$

where X_(C) is the fluorescence signal from the compound-treated well; M_(B) is the average fluorescence signal from puromycin-treated wells; M_(D) is the average fluorescence signal from DMSO-treated wells. The anti-HCV replication activity was determined by the luminescence signal generated from the reporter renilia luciferase of the HCV replicon. The percent inhibition on HCV replicon was calculated using equation 1, where X_(C) is the luminescence signal from compound-treated well; M_(B) average luminescence signal from the ITMM-191-treated wells; M_(D) is the average luminescence signal from DMSO-treated wells.

The CC₅₀ values ware determined as the testing compound concentration that caused a 50% decrease of cell viability. The EC₅₀ values were determined as the testing compound concentration that caused a 50% decrease in HCV replication. Both CC₅₀ and EC₅₀ values were obtained using Pipeline Pilot 5.0 software package (Accelrys, San Diego, Calif.) by nonlinear regression fitting of experimental data to equation 2:

$\begin{matrix} {y = {d + \frac{a - d}{\left\lbrack {1 + \left( \frac{x}{c} \right)^{b}} \right\rbrack}}} & (2) \end{matrix}$

where y is the observed % inhibition of HCV replicon at x concentration of compound; d is estimated response at zero compound concentration; a is estimated response at infinite compound concentration; c Is the mid-range concentration (CC₅₀ or EC₅₀); b is the Hill slope factor.

The combination study experimental data were analyzed using the MacSynergy II program developed by Prichard and Shipman (Prichard M M, Aseltine K R, Shipman C, Jr. MacSynergy™ II, Version 1.0. University of Michigan, Ann Arbor, Mich., 1993). The software (MacSynergy™ II, University of Michigan, Mich.) calculates theoretical inhibition assuming an additive interaction between drugs (based on the Bliss Independence model) and quantifies statistically significant differences between the theoretical and observed inhibition values. Plotting these differences in three dimensions results in a surface where elevations in the Z-plane represent antiviral synergy and depressions represent antiviral antagonism between compounds. The calculated volumes of surface deviations are expressed in μM²%. Per Prichard and Shipman, combination effects are defined as:

-   Strong synergy: >100 μM²% -   Moderate synergy: >50 and ≧100 μM²% -   Minor synergy: >25 and ≦50 μM²% -   Additivity: ≦25 and >−25 μM²% -   Minor antagonism: ≦−25 and >−50 μM²% -   Moderate antagonism: ≦−50 and >−100 μM²% -   Strong astagonism: ≦−100 μM²%

For eash combination study, three independent experiments were performed with four replicates in each experiment

Results Antiviral Activity and Cytotoxicity of Individual Compounds in HCV Genotype 1a Replicon Assay

The anti-HCV activity and cytotoxicity of Compound 10 combined with other anti-HCV compounds were tested in Huh-7 cells carrying an HCV genotype 1a replicon. The EC₅₀ and CC80 values for ail compounds are listed in the following Table. There is no significant cytotoxicity observed for ail individual compounds up to the highest concentrations tested in the combination assay.

TABLE 8.1 EC₅₀ and CC₅₀ of Compounds used in this Study against HCV Genotype 1a Replicon Compounds Class EC₅₀ ^(a) (nM) CC₅₀ ^(a) (nM) Compound 10 NS5B Nucleoside Prodrug 39 >82446 Compound 6 NS5A Inhibitor 0.032 >44400 Compound 1 NS5B Non-nucleoside 18 >44400 Compound 5 NS5B Non-nucleoside 14 >44400 Compound 3 NS3 Protease Inhibitor 46 >22200 RBV Nucleoside Analog 33626 >88800 ^(a)Values are geometric means for three or more independent experiments Antiviral Activity and Cytotoxicity of Compound 10 in Combination with Other Classes of Anti-HCV Agents.

The antiviral effects of Compound 10 in combination with other anti-HCV compounds were evaluated using the HCV genotype 1a replicon. The results were analyzed using MacSynergy II, which provides surface plots displaying significant deviations from additivity. Synergy and antagonism volumes (μM²%) calculated from deviations from additive surface are summarised in the following Table. At 95% confidence interval, the mean synergy and antagonism volumes were between 25 and −25 μM2 % when Compound 10 was combined with Compound 6, Compound 1, Compound 5, or Compound 3 indicative of additive interaction with Compound 10. Furthermore, Compound 10 shows a synergy volume in the range of 20 to 50 μM²% when combined with RBV, suggesting a minor synergistic interaction. In combination studies using direct acting antivirals with Compound 10, cell viability was greater than 93% at the highest concentrations of compound combinations tested while studies analyzing the combined effects of Compound 10 and RBV showed cell viability greater than 85% at the highest combined drug concentrations.

TABLE 8.2 Quantification of Antiviral Synergy and Antagonism and Drug Interactions for Drug Combinations with Compound 10 Compound Used in Combination Synergy Antagonism with Volume Volume Compound 10 Class (nM²)^(a) (nM²)^(a) Interaction Compound 6 NS5A 3.3 ± 4.2  −7.7 ± 13.3 Additive Inhibitor Compound 1 NS5B Non- 4.7 ± 8.1 −11.7 ± 10.0 Additive nucleoside Compound 5 NS5B Non- 1.3 ± 2.3 −5.7 ± 9.0 Additive nucleoside Compound 3 NS3 Protease 1.0 ± 1.7 −3.0 ± 4.4 Additive Inhibitor RBV Nucleoside 35.3 ± 3.2  −2.0 ± 2.0 Minor Analog synergy ^(a)Values represent the mean ± standard deviation of three independent experiments performed in four replicates

TABLE 8.3 Quantification of Cytotoxicity In Compound Combinations Highest Concentration Cytotoxicity of Compound at Highest Used With Concentration Highest of Compound Compound Used in Concentration Combinations Combination (320 nM) of (% inhibition with Compound 10 Compound 10 on cell growth) Compound 6 0.16 nM  5.0 ± 5.0 Compound 1 120 nM 7.0 ± 4.6 Compound 5  64 nM 4.3 ± 2.9 Compound 3 240 nM 2.0 ± 3.5 RBV 16000 nM  14.0 ± 4.4  ^(a)Values represent the mean ± standard deviation of three independent experiments performed in four replicates

Conclusions

The antiviral activity of Compound 10 was tested in combination with Compound 6, Compound 1, Compound 5, Compound 3, or RBV. Compound 10 showed additive antiviral activity in combination with Compound 6, Compound 1, Compound 5, or Compound 3, and minor synergy with RBV.

In summary, these findings sopped the potential of Compound 10 to be used in combination with Compound 6, Compound 1, Compound 5, Compound 3 or RBV to achieve enhanced viral suppression without reducing the efficacy of any of the individual drugs.

Clinical Example 1 Clinical Testing of Anti-HCV Activity of the Combination of Compound 1 and Compound 2

This Clinical Example shows that the combination of Compound 1 and Compound 2 plus ribavirin is more effective at reducing HCV viral load, and suppressing HCV viral rebound, then the combination of Compound 1 plus Compound 2 without ribavirin.

Clinical Trial Design

A Phase 2, randomized, open-label trial of Compound 2 plus Compound 1 alone and in combination with ribavirin for 28 days in treatment-naive subjects with chronic genotype 1 HCV infection. Subjects in Arm 1 received Compound 2 at 75 mg+Compound 1 at 40 mg, both administered twice daily (BID) (double regimen) and subjects in Arm 2 received Compound 2 at 75 mg+Compound 1 at 40 mg, both administered BID, and plus ribavirin, also administered BID (triple regimen) for 28 days.

On Day 28, all subjects were to initiate PEG/Ribavirin. Additionally, the protocol called for subjects with an insufficient virologic response (<2 log₁₀ IU/mL reduction from baseline HCV RNA by Day 5) or virologic rebound (HCV RNA increase of >0.5 log₁₀ IU/mL from nadir confirmed over two time points occurring after Day 5 with an absolute value>1000 IU/mL) to initiate PEG/RIBA prior to Day 28.

For subjects with insufficient virologic response, the combination of pegylated interferon (PEG) and ribavirin (RIBA) was initiated prior to Day 28 with or without continuation Compound 2+Compound 1. As a result, by Day 28 of the study, subjects were receiving one of four

-   -   (i) Compound 2+Compound 1,     -   (ii) Compound 2+Compound 1+RIBA,     -   (iii) Compound 2+Compound 1+PEG/RIBA, or     -   (iv) PEG/RIBA.

A total of 31 subjects were enrolled and started dosing (16 subjects received the double regimen in Arm 1 and 15 subjects received the triple regimen in Arm 2). Preliminary subject demographics and baseline characteristics (Table XVI) were generally comparable between Arms 1 and 2, aside from a greater number of subjects with genotype 1b in Arm 2. Four subjects were identified as HCV genotype 1b at screening (one subject on the dual regimen and three subjects on the triple regimen), but have not been confirmed as genotype 1a or 1b upon further analysis, with further assessment ongoing.

No subjects have experienced serious adverse events. Study medications have been generally well-tolerated, with all adverse events being Grade 1-2 in severity, except for a single Grade 3 fatigue, which was the only treatment emergent adverse event leading to study drug discontinuation. Prior to the initiation of PEG/Ribavirin, the most common treatment-emergent adverse events occurring in more than one subject were headache (n=5), and diarrhea or nausea (n=3 each) in Arm 1 and headache (n=7), diarrhea or fatigue (n=3 each), nausea, asthenia, pruritis or insomnia (n=2 each) in Arm 2. When Compound 2+Compound 1 were given in combination with PEG/RIBA, the only adverse events occurring in more then one subject were influenza-like illness (n=5) and myalgia (n=3). both common adverse events with PEG/RIBA therapy. With regard to laboratory abnormalities, there were no Grade 4 events during the 28-day treatment period. Among subjects receiving the study drugs, there were two treatment-emergent Grade 3 elevations in total bilirubin in the ribavirin containing Arm 2 (occurring at Day 7, but resolving with continued dosing of study drug). There were also 2 Grade-1 elevations and a single Grade-2 elevation in total bilirubin among other subjects in this dosing Arm (with ribavirin). Among subjects in Arm-1 (no ribavirin), there were four Grade-1total bilirubin elevations. ALT values were reduced approximately 40 U/L from baseline in both arms by Day 14. Median GTcF was not significantly changed from baseline in either study arm and no subjects discontinued study drugs due to QTc abnormalities. Preliminary safety data are summarized in Table XVIII.

Plasma HCV RNA was monitored approximately twice weekly to gauge viroiegic response in relation to the protocol-specified criteria for early initiation of PEG/RIBA. From preliminary analysis of the HCV RNA values, the median maximal decline in HCV RNA was 3.9 log₁₀ IU/mL for the dual regimen and 5.0 log₁₀ IU/mL for the triple regimen. The median time to maximal decline in HCV RNA was 7 days for the dual regimen and 14 days for the triple regimen, with the difference attributed to delayed incidence and onset of viral breakthrough in the ribavirin containing arm. Three of 15 (20%) subjects receiving the dual regimen and 10 of 13 (77%) subjects receiving the triple regimen had nadir HCV RNA values≦30 IU/mL (excluding non-GT1 subjects). 13/16 (81%) subjects receiving Compound 2/Compound 1 and 6/15 (40%) subjects receiving Compound 2/Compound1 /Ribavirin initiated PEG or PEG/RIbavirin prior to the scheduled start on Day 28 of the study. Additional details of virologie outcomes are provided in

Results. Compound 2+Compound 1 alone and in combination with RIBA were well-tolerated for up to 28 days by HCV subjects in this study, both before and following the addition of PEG or PEG/Ribavirin. Both regimens yielded potent suppression of HCV RNA, with greater and more sustained activity in the three drug regimen.

TABLE XVI Preliminary Subject Demographics and Baseline Characteristics Arm #2: Arm #1: Compound 2 at Compound 2 at 75 mg BID + 75 mg BID + Compound 1 Compound 1 at at 40 mg 40 mg BID BID + RIBA (n = 16) (n = 15) Age in years - 47 55 Median (range) (30, 66) (27, 63) Sex 14 male 11 male  2 female  4 female Ethnicity 16 Non-Hispanic/ 15 Non-Hispanic/ Latino Latino Race 13 White 13 White  2 Black  2 Black  1 Asian  0 Asian Baseline Weight in 86.1 79.0 kg - Median (range) (57.8, 110.5) (51, 127.5) Baseline BMI in 27.1 24.7 kg/M² - Median (range) (21.5, 34.1) (19.9, 37.6) Baseline Log₁₀ HCV  6.17  6.34 RNA (IU/mL) from Central (5.25, 7.26) (5.41, 7.19) lab- Median (range) Central lab Baseline HCV  8 1a  3 1a Genotype  8 1b 12 1b

TABLE XVII Preliminary Safety Results Arm 1: Arm 2: Compound 2 at 75 mg Compound 2 at 75 mg BID + BID + Compound 1 at Compound 1 at 40 mg 40 mg BID BID + RIBA (n = 16) (n = 15) Grade 3 Adverse Events (AEs): Fatigue 1 0 Grade 1/ Grade 2 (AEs): Headache 5 (31%) 7 (47%) Diarrhea 3 (19%) 3 (20%) Nausea 3 (19%) 2 (13%) Fatigue 0 3 (20%) Asthenia 0 2 (13%) Pruritia 1 (6%)  2 (13%) Insomnia 0 2 (13%) Grade 3 Laboratory Abnormalities: Bilirubin 0 2 Grade 1/ Grade 2 Laboratory Abnormalities: Bilirubin 4 3 Hemoglobin 0 2 Glucose 8 5 (nonfasting)

TABLE XVIII Preliminary Virologic Outcomes Arm 2: Arm 1: Compound 2 at Compound 2 at 75 mg BID + 75 mg BID + Arm 2: Compound 1 at Arm 1: Compound 1 at Compound 2 at 40 mg BID + Compound 2 at 40 mg BID 75 mg BID + Ribavirin 75 mg BID + Unconfirmed Compound 1 at Unconfirmed Compound 1 at GT1 Subjects 40 mg BID + GT1 Subjects 40 mg BID Excluded Ribavirin Excluded (n = 16) (n = 15)* (n = 15) (n = 13) Median maximal −3.9 log₁₀ IU/mL −4.0 log₁₀ IU/mL −5.0 log₁₀ IU/mL −5.0 log₁₀ IU/mL HCV RNA decline Mean maximal −3.4 log₁₀ IU/mL −3.6 log₁₀ IU/mL −4.5 log₁₀ IU/mL −4.9 log₁₀ IU/mL HCV RNA decline Mean time to 16 days 16 days 23 days 23 days Breakthrough Subjects with 3/16 (19%) 3/15 (20%) 10/15 (63%)  10/13 (77%)  HCV RNA nadir <50 IU/mL Subjects with   12 (75%) 12/15 (80%)  6/15 (40%) 6/13 (46%) Breakthrough** Day 28 Response: RVR at <25 IU/mL 1/16 (6%) 1/15 (7%) 5/15 (33%) 5/13 (38%) RVR at <50 IU/mL 1/16 (6%) 1/15 (7%) 6/15 (40%) 6/13 (46%) *GT1 is an abbreviation for HCV Genotype 1. Subjects 1011, 1012, and 1043 at one French study center were excluded; Subject 1004 was not excluded **Breakthrough defined as >1 log increase in HCV RNA above nadir value or HCV RNA >25 IU/mL following a nadir of <25 IU/mL

The data presented in Table XVIII shew that there was an approximately 10 fold greater decline in both the median maximal HCV RNA level and the mean maximal HCV RNA level in response to the combination of Compound 2+Compound 1 in the presence of dbavidn compared to the absence of ribavirin. Also, the number of study subjects having an HCV RNA nadir below 50 IU/mL is greater in the presence of ribavirin than in the absence of ribavirin. These results show that ribavirin, in the absence of interferon, significantly potentiates the antiviral activity of the combination of Compound 1 and Compound 2.

Additionally, the mean time to HCV breakthrough, which is a measure of the eventually increase in HCV viral load as the virus mutates and becomes less susceptible to the antiviral drugs, is greater in the presence of ribavirin than in the absence of ribavirin. Further, the number of subjects showing viral breakthrough is substantially less in the presence of ribavirin than in the absence of ribavirin. These results show that the HCV virus is less able to develop resistance to the combination of Compound 1 and Compound 2 in the presence of ribavirin.

Further, the data presented in Table XVIII shows that the number of patients achieving a Rapid Viroiogic Response (RVR) in the presence of ribavirin is significantly greater than in the absence of ribavirin. Achievement of RVR positively correlates with cure of HCV infection.

Taken together the data presented in Table XVIII show that the combination of Compound 1, Compound 2, and ribavirin causes a rapid and clinically significant reduction in HCV viral load, with a reduced viral rebound, even in the absence of administration of interferon.

Although specific embodiments of the present invention are herein illustrated and described in detail, the invention is not limited thereto. The above detailed descriptions are provided as exemplary of the present invention and should not be construed as constituting any limitation of the invention. Modifications will be obvious to those skilled in the art, and ait modifications that do not depart from the spirit of the invention ara intended to be included with the scope of the appended claims. 

What is claimed is:
 1. A composita comprising: 1) compound 10 or a pharmaceutically acceptable salt thereof and 2) compound 5 or a pharmaceutically acceptable salt thereof, or compound 6 or a pharmaceutically acceptable salt thereof.
 2. The composition of claim 1 which composes: 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 5 or a pharmaceutically acceptable salt thereof.
 3. The composition of claim 1 which composes: 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 8 or a pharmaceutically acceptable salt thereof.
 4. The composition of claim 1 which comprises: 1) compound 10 or a pharmaceutically acceptable salt thereof, 2) compound 5 or a pharmaceutically acceptable salt thereof, and 3) compound 6 or a pharmaceutically acceptable salt thereof.
 5. The composition of any one of claims 1-4 which further comprises one or more pharmaceutically acceptable diluents or carriers.
 6. The composition of claim 5 which is formulated as a unit dosage form for once daily administration.
 7. The composition of any one of claims 5-8 which is formulated for oral administration.
 8. The composition of any one of claims 5-7 which is formulated, as a tablet.
 9. The composition of any one of claims 1-8 which further comprises ribavirin.
 10. A method of treating an HCV infection in a human, comprising administering to the human: 1) a compound 10 or a pharmaceutically acceptable salt thereof and 2) a compound 5 or a pharmaceutically acceptable salt thereof, or compound 8 or a pharmaceutically acceptable salt thereof.
 11. The method of claim 10 wherein: 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) a compound 5 or a pharmaceutically acceptable salt thereof are administered.
 12. The method of claim 10 wherein: 1) a compound 10 or a pharmaceutical acceptable salt thereof, and 2) a compound 8 or a pharmaceutically acceptable salt thereof are administered.
 13. The method of claim 10 wherein: 1) a compound 10 or a pharmaceutically acceptable salt thereof, 2) a compound 5 or a pharmaceutically acceptable salt thereof, and 3) a compound 8 or a pharmaceutically acceptable salt thereof are administered.
 14. The method of claim 10 wherein a composition as described in any one of claims 1-8 is administered to the human.
 15. A method of ameliorating one or more symptoms of an HCV infection in a human, comprising administering to the human: 1) a compound 10 or a ptorniaceuficaliy acceptable salt thereof and 2) a compound 5 or a pharmaceutically acceptable salt thereof, or compound 8 or a pharmaceutically acceptable salt thereof.
 16. The method of claim 15 wherein: 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) a compound 5 or a pharmaceutically acceptable salt thereof are administered.
 17. The method of claim 15 wherein: 1) a compound 10 or a pharmaceutically acceptable salt thereof, and 2) a compound or a pharmaceutically acceptable salt thereof are administered.
 18. The method of claim 15 wherein: 1) a compound 10 or a pharmaceutically acceptable salt thereof, 2) a compound 5 or a pharmaceutically acceptable salt thereof, and 3) a compound 6 or a pharmaceutically acceptable salt thereof are administered.
 19. The method of claim 15 wherein a composition as described in any one of claims 1-8 is administered to the human.
 20. A method of reducing viral load in a human with HCV, comprising administering to the human: 1) a compound 10 or a pharmaceutically acceptable salt thereof and 2) a compound 5 or a pharmaceutically acceptable salt thereof, or compound 6 or a pharmaceutically acceptable salt thereof.
 21. The method of claim 20 wherein: 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) a compound 5 or a pharmaceutically acceptable salt thereof are administered.
 22. The method of claim 20 wherein: 1) a compound 10 or a pharmaceutically acceptable salt thereof, and 2) a compound or a pharmaceutically acceptable salt thereof are administered,
 23. The method of claim 20 wherein: 1) a compound 10 or a pharmaceutically acceptable salt thereof, 2) a compound 5 or a pharmaceutical acceptable salt thereof, and 3) a compound 6 or a pharmaceutically acceptable salt thereof are administered.
 24. The method of claim 20 wherein a composition as described in any one of claims 1-8 is administered to the human.
 25. A method for reducing emergence of HCV quaslspecles with resistance to coadministered oral antiviral agents in a human, comprising administering to the human: 1) a compound 10 or a pharmaceutically acceptable salt thereof and 2) a compound 5 or a pharmaceutically acceptable salt thereof, or compound 6 or a pharmaceutically acceptable salt thereof.
 20. The method of claim 25 wherein; 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) a compound 5 or a pharmaceutically acceptable salt thereof are administered.
 27. The method of claim 25 wherein: 1) a compound 10 or a pharmaceutically acceptable salt thereof, and 2) a compound 8 or a pharmaceutically acceptable salt thereof are administered.
 28. The method of claim 25 wherein: 1) a compound 10 or a pharmaceutically acceptable salt thereof, 2) a compound 5 or a pharmaceutically acceptable salt thereof, and 3) a compound 8 or a pharmaceutically acceptable salt thereof are administered.
 29. The method of claim 25 wherein a composition as described in any one of claims 1-3 is administered to the human.
 30. The method of any one of claims 10-29 wherein an interferon is not administered to the human.
 31. The method of any one of claims 10-29 further comprising administering ribavirin to the human.
 32. The method of any one of claims 10-29 further comprising administering one or more additional agents selected from ribavirin, an interferon, alpha-glucosidase 1 inhibitors, hepatoprotectants, TLR-7 agonists, cyclophilin inhibitors, HCV viral entry inhibitors, HCV maturation inhibitors, and HCV IRES inhibitors fo the human.
 33. A composition as described in any one of claims 1-9 for use in medical therapy.
 34. A composition as described in any one of claims 1-9 for the prophylactic or therapeutic treatment of an HCV infection.
 35. The use of a composition as described in any one of claims 1-9 to prepare a medicament for treating an HCV infection in a human,
 36. The use of a composition as described in any one of claims 1-9 to prepare a medicament for ameliorating one or more symptoms of an HCV infection in a human.
 37. A composition as described in any one of claims 1-9 for reducing viral load.
 38. The use of a composition as described in any one of claims 1-9 to prepare a medicament for reducing viral load in a human,
 39. A composition as described in any one of claims 1-9 for reducing emergence of HCV quasispedes with resistance to coadministered oral antiviral agents.
 40. The use of a composition as described in any one of ciaims 1-9 to prepare a medicament for reducing emergence of HCV quasispedes with resistance to coadministered oral antiviral agents in a human.
 41. The composition of claim 34, 37, or 39 which is not for administration with an interferon.
 42. The composition of claim 34, 37, or 39 which is for administration with ribavirin.
 43. The composition of claim 34, 37, or 39 which is for administration with one or more additional agents selected from ribavirin, an interferon, alpha-glucosidase 1 inhibitors, hepatoprotectents, TLR-7 agonists, cyclophin inhibitors, HCV viral entry inhibitors, HCV maturation inhibitors, and HCV IRES inhibitors.
 44. The use of any one of claims 35,36, 38, and 40 wherein the medicament is not for administration with an interferon.
 45. The use of any one of claims 35,36, 38, and 40 wherein the medicament is for administration with ribavirin.
 46. The use of any one of claims 35,38, 38, and 40 wherein the medicament is for administration with one or more additional agents selected from ribavirin, an interferon, alpha-glucosidase 1 inhibitors, hepatoprotectants, TLR-7 agonists, cyclophilin inhibitors, HCV viral entry inhibitors, HCV maturation inhibitors, and HCV IRES inhibitors. 